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Chapter 9
Organization of the
Human Genome
9
KEY CONCEPTS
•
The human genome is subdivided into a large nuclear genome with more than 26,000
genes, and a very small circular mitochondrial genome with only 37 genes. The nuclear
genome is distributed between 24 linear DNA molecules, one for each of the 24 different
types of human chromosome.
•
Human genes are usually not discrete entities: their transcripts frequently overlap those
from other genes, sometimes on both strands.
•
Duplication of single genes, subchromosomal regions, or whole genomes has given rise to
families of related genes.
•
Genes are traditionally viewed as encoding RNA for the eventual synthesis of proteins, but
many thousands of RNA genes make functional noncoding RNAs that can be involved in
diverse functions.
•
Noncoding RNAs often regulate the expression of speciﬁc target genes by base pairing with
their RNA transcripts.
•
Some copies of a functional gene come to acquire mutations that prevent their expression.
These pseudogenes originate either by copying genomic DNA or by copying a processed
RNA transcript into a cDNA sequence that reintegrates into the genome
(retrotransposition).
•
Occasionally, gene copies that originate by retrotransposition retain their function because
of selection pressure. These are known as retrogenes.
•
Transposons are sequences that move from one genomic location to another by a cut-andpaste or copy-and-paste mechanism. Retrotransposons make a cDNA copy of an RNA
transcript that then integrates into a new genomic location.
•
Very large arrays of high-copy-number tandem repeats, known as satellite DNA, are
associated with highly condensed, transcriptionally inactive heterochromatin in human
chromosomes.
256
Chapter 9: Organization of the Human Genome
1.1% ~4%
<2%
~32%
~44%
~45%
~66%
~6.5%
mitochondrial genome
nuclear genome
highly conserved sequences
poorly conserved sequences
protein-coding genes
transposon-based repeats
RNA genes, regulatory sequences
heterochromatin
other sequences
The human genome comprises two parts: a complex nuclear genome with more
than 26,000 genes, and a very simple mitochondrial genome with only 37 genes
(Figure 9.1). The nuclear genome provides the great bulk of essential genetic
information and is partitioned between either 23 or 24 different types of chromosomal DNA molecule (22 autosomes plus an X chromosome in females, and an
additional Y chromosome in males).
Mitochondria possess their own genome—a single type of small circular
DNA—encoding some of the components needed for mitochondrial protein synthesis on mitochondrial ribosomes. However, most mitochondrial proteins are
encoded by nuclear genes and are synthesized on cytoplasmic ribosomes before
being imported into the mitochondria.
As detailed in Chapter 10, sequence comparisons with other mammalian
genomes and vertebrate genomes indicate that about 5% of the human genome
has been strongly conserved during evolution and is presumably functionally
important. Protein-coding DNA sequences account for just 1.1% of the genome.
The other 4% or so of strongly conserved genome sequences consists of nonprotein-coding DNA sequences, including genes whose ﬁnal products are functionally important RNA molecules, and a variety of cis-acting sequences that
regulate gene expression at DNA or RNA levels. Although sequences that make
non-protein-coding RNA have not generally been so well conserved during evolution, some of the regulatory sequences are much more strongly conserved than
protein-coding sequences.
Protein-coding sequences frequently belong to families of related sequences
that may be organized into clusters on one or more chromosomes or be dispersed
throughout the genome. Such families have arisen by gene duplication during
evolution. The mechanisms giving rise to duplicated genes also give rise to nonfunctional gene-related sequences (pseudogenes).
One of the big surprises in the past few years has been the discovery that the
human genome is transcribed to give tens of thousands of different noncoding
RNA transcripts, including whole new classes of tiny regulatory RNAs not previously identiﬁed in the draft human genome sequences published in 2001.
Although we are close to obtaining a deﬁnitive inventory of human protein-coding genes, our knowledge of RNA genes remains undeveloped. It is abundantly
clear, however, that RNA is functionally much more versatile than we previously
suspected. In addition to a rapidly increasing list of human RNA genes, we have
also become aware of huge numbers of pseudogene copies of RNA genes.
A very large fraction of the human genome, and other complex genomes, is
made up of highly repetitive noncoding DNA sequences. A sizeable component
is organized in tandem head-to-tail repeats, but the majority consists of interspersed repeats that have been copied from RNA transcripts in the cell by reverse
Figure 9.1 Sequence conservation and
sequence classes in the human nuclear
and mitochondrial genomes. To get an
idea of the vast diﬀerence in scale between
the nuclear (left) and mitochondrial
(right) genomes, the tiny red dot in the
center represents the equivalent of
25 mitochondrial DNA (mtDNA) genomes on
the same scale as the single nuclear genome
on the left. Note also the profound diﬀerence
between the two genomes in the fractions
of highly conserved DNA and also in the
fraction of highly repetitive noncoding DNA.
GENERAL ORGANIZATION OF THE HUMAN GENOME
257
transcriptase. There is a growing realization of the functional importance of such
repeats.
In this chapter we primarily consider the architecture of the human genome.
We outline the different classes of DNA sequence, describe brieﬂy what their
function is, and consider how they are organized in the human genome. In later
chapters we describe other aspects of the human genome: how it compares with
other genomes, and how evolution has shaped it (Chapter 10), DNA sequence
variation and polymorphism (Chapter 13), and aspects of human gene expression (Chapter 11).
9.1 GENERAL ORGANIZATION OF THE HUMAN
GENOME
The DNA sequence of the human mitochondrial genome was published in 1981,
and a detailed understanding of how mitochondrial DNA (mtDNA) works has
been built up since then. The more complex nuclear genome has been a much
more formidable challenge. Comprehensive sequencing of the nuclear genome
began in the latter part of the 1990s, and by 2004 essentially all of the euchromatic portion of the genome had been sequenced. Our knowledge of the nuclear
genome remains fragmentary, however. As we see below, we still do not know
how many genes there are in the nuclear genome, and recently obtained data are
radically changing our perspective on how it is organized and expressed.
The mitochondrial genome is densely packed with genetic
information
The human mitochondrial genome consists of a single type of circular doublestranded DNA that is 16.6 kilobases in length. The overall base composition is
44% (G+C), but the two mtDNA strands have signiﬁcantly different base compositions: the heavy (H) strand is rich in guanines, but the light (L) strand is rich in
cytosines. Cells typically contain thousands of copies of the double-stranded
mtDNA molecule, but the number can vary considerably in different cell types.
During zygote formation, a sperm cell contributes its nuclear genome, but
not its mitochondrial genome, to the egg cell. Consequently, the mitochondrial
genome of the zygote is usually determined exclusively by that originally found in
the unfertilized egg. The mitochondrial genome is therefore maternally inherited: males and females both inherit their mitochondria from their mother, but
males do not transmit their mitochondria to subsequent generations. During
mitotic cell division, the multiple mtDNA molecules in a dividing cell segregate
in a purely random way to the two daughter cells.
H strand
7S DNA
Replication of mitochondrial DNA
The replication of both the H and L strands is unidirectional and starts at speciﬁc
origins. Although the mitochondrial DNA is principally double-stranded, repeat
synthesis of a small segment of the H-strand DNA produces a short third DNA
strand called 7S DNA. The 7S DNA strand can base-pair with the L strand and
displace the H strand, resulting in a triple-stranded structure (Figure 9.2). This
region contains many of the mtDNA control sequences (including the major promoter regions) and so it is referred to as the CR/D-loop region (where CR denotes
control region, and D-loop stands for displacement loop).
The origin of replication for the H strand lies in the CR/D-loop region, and
that of the L strand is sandwiched between two tRNA genes (Figure 9.3). Only
after about two-thirds of the daughter H strand has been synthesized (by using
the L strand as a template and displacing the old H strand) does the origin for
L-strand replication become exposed. Thereafter, replication of the L strand proceeds in the opposite direction, using the H strand as a template.
Mitochondrial genes and their transcription
The human mitochondrial genome contains 37 genes, 28 of which are encoded
by the H strand and the other nine by the L strand (see Figure 9.3). Whereas
nuclear genes often have their own dedicated promoters, the transcription of
mitochondrial genes resembles that of bacterial genes. Transcription of mtDNA
L strand
Figure 9.2 D-loop formation in
mitochondrial DNA. The mitochondrial
genome is not a simple double-stranded
circular DNA. Repeat synthesis of a small
segment of the H (heavy) strand results in a
short third strand (7S DNA), which can basepair with the L (light) strand and so displace
the H strand to form a local triple-stranded
structure that contains many important
regulatory sequences and is known as the
CR/D-loop region (shown by shading and in
enlarged form for clarity).
258
Chapter 9: Organization of the Human Genome
OH P
H
7S
DNA
Phe
Thr
H
strand
CYB
12S
Val
Pro
S
16
Glu
PL
ND6
Leu
ND5
ND1
OL
ND4
L
strand
Ile
f-Met
Gln
Leu
Ser
His
Ala
Asn
ND2
Cys
Tyr
ND4L
Arg
ND3
Gly
Trp
Ser
CO1
CO3
ATP6 ATP8
CO2
Asp
Lys
replication origins and
OHOL direction of synthesis
of H and L strands
rRNA genes
promoters and
PHPL direction of transcription
of H and L strands
genes encoding proteins
tRNA genes
starts from common promoters in the CR/D-loop region and continues round
the circle (in opposing directions for the two different strands), to generate large
multigenic transcripts. The mature RNAs are subsequently generated by cleavage
of the multigenic transcripts.
Almost two-thirds (24 out of 37) of the mitochondrial genes specify a functional noncoding RNA as their ﬁnal product. There are 22 tRNA genes, one for
each of the 22 types of mitochondrial tRNA. In addition, two rRNA genes are dedicated to making 16S rRNA and 12S rRNA (components of the large and small
subunits, respectively, of mitochondrial ribosomes). The remaining 13 genes
encode polypeptides, which are synthesized on mitochondrial ribosomes. These
13 polypeptides form part of the mitochondrial respiratory complexes, the
enzymes of oxidative phosphorylation that are engaged in the production of ATP.
However, the great majority of the polypeptides that make up the mitochondrial
oxidative phosphorylation system plus all other mitochondrial proteins are
encoded by nuclear genes (Table 9.1). These proteins are translated on cytoplasmic ribosomes before being imported into the mitochondria.
Unlike its nuclear counterpart, the human mitochondrial genome is extremely
compact: all 37 mitochondrial genes lack introns and are tightly packed (on average, there is one gene per 0.45 kb). The coding sequences of some genes (notably
those encoding the sixth and eighth subunits of the mitochondrial ATP synthase)
show some overlap (Figure 9.4) and, in most other cases, the coding sequences of
neighboring genes are contiguous or separated by one or two noncoding bases.
Some genes even lack termination codons; to overcome this deﬁciency, UAA
codons have to be introduced at the post-transcriptional level (see Figure 9.4).
The mitochondrial genetic code
Prokaryotic genomes and the nuclear genomes of eukaryotes encode many hundreds to usually many thousands of different proteins. They are subject to a universal genetic code that is kept invariant: mutations that could potentially change
Figure 9.3 The organization of the human
mitochondrial genome. The H strand
is transcribed from two closely spaced
promoter regions ﬂanking the tRNAPhe
gene (grouped here as PH); the L strand is
transcribed from the PL promoter in the
opposite direction. In both cases, large
primary transcripts are produced and
cleaved to generate RNAs for individual
genes. All genes lack introns and are closely
clustered. The symbols for protein-coding
genes are shown here without the preﬁx
MT- that signiﬁes mitochondrial gene. The
genes that encode subunits 6 and 8 of the
ATP synthase (ATP6 and ATP8) are partly
overlapping. Other polypeptide-encoding
genes specify seven NADH dehydrogenase
subunits (ND4L and ND1–ND6), three
cytochrome c oxidase subunits (CO1–CO3),
and cytochrome b (CYB). tRNA genes are
represented with the name of the amino
acid that they bind. The short 7S DNA strand
is produced by repeat synthesis of a short
segment of the H strand (see Figure 9.2).
For further information, see the MITOMAP
database at http://www.mitomap.org/.
GENERAL ORGANIZATION OF THE HUMAN GENOME
259
TABLE 9.1 THE LIMITED AUTONOMY OF THE MITOCHONDRIAL GENOME
Mitochondrial component
Encoded by
Mitochondrial
genome
Nuclear genome
Components of oxidative phosphorylation
system
13 subunits
80 subunits
I
NADH dehydrogenase
7
42
II
Succinate CoQ reductase
0
4
III
Cytochrome b–c1 complex
1
10
IV
Cytochrome c oxidase complex
3
10
V
ATP synthase complex
2
14
Components of protein synthesis apparatus
24 RNAs
79 proteins
rRNA
2
0
tRNA
22
0
Ribosomal proteins
0
79
Other mitochondrial proteins
0
Alla
aIncludes mitochondrial DNA and RNA polymerases plus numerous other enzymes, structural and
transport proteins, etc.
the genetic code are likely to produce at least some critically misfunctional proteins and so are strongly selected against. However, the much smaller mitochondrial genomes make very few polypeptides. As a result, the mitochondrial genetic
code has been able to drift by mutation to be slightly different from the universal
genetic code.
In the mitochondrial genetic code there are 60 codons that specify amino
acids, one fewer than in the nuclear genetic code. There are four stop codons:
UAA and UAG (which also serve as stop codons in the nuclear genetic code) and
AGA and AGG (which specify arginine in the nuclear genetic code; see Figure
1.25). The nuclear stop codon UGA encodes tryptophan in mitochondria, and
AUA speciﬁes methionine not isoleucine.
The mitochondrial genome speciﬁes all the rRNA and tRNA molecules needed
for synthesizing proteins on mitochondrial ribosomes, but it relies on nuclearencoded genes to provide all other components, such as the protein components
of mitochondrial ribosomes and aminoacyl tRNA synthetases. Because there are
only 22 different types of human mitochondrial tRNA, individual tRNA molecules
need to be able to interpret several different codons. This is possible because of
third-base wobble in codon interpretation. Eight of the 22 tRNA molecules have
anticodons that each recognize families of four codons differing only at the third
base. The other 14 tRNAs recognize pairs of codons that are identical at the ﬁrst
two base positions and share either a purine or a pyrimidine at the third base.
Between them, therefore, the 22 mitochondrial tRNA molecules can recognize a
total of 60 codons [(8 ¥ 4) + (14 ¥ 2)].
MT-ATP8
8366
1
Met
ATG
8577 9202 9206
8522
53
68
Pro Lys Trp Thr Lys Ile Cys Ser Leu His Ser Leu Pro Pro Gln Ser Stop
CCAAAATGAACGAAAATCTGTTCGCTTCATTCATTGCCCCCACAATCCTAGGCCTA
Met Asn Glu Asn Leu Phe Ala Ser Phe Ile Ala Pro Thr Ile Leu Gly Leu
1
17
MT-ATP6
ACATA
Thr
226
Figure 9.4 The MT-ATP6 and MT-ATP8
genes are transcribed in diﬀerent
reading frames from overlapping
segments of the mitochondrial H
strand. MT-ATP8 is transcribed from
nucleotides 8366 to 8569, and MT-ATP6
from 8527 to 9204. After transcription,
the RNA encoding the ATP synthase 6
subunit is cleaved after position 9206
and polyadenylated, resulting in a
C-terminal UAA codon where the ﬁrst
two nucleotides are derived ultimately
from the TA at positions 9205–9206
and the third nucleotide is the ﬁrst A of
the poly(A) tail.
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Chapter 9: Organization of the Human Genome
TABLE 9.2 THE HUMAN NUCLEAR AND MITOCHONDRIAL GENOMES
Nuclear genome
Mitochondrial genome
Size
3.1 Gb
16.6 kb
Number of diﬀerent DNA molecules
23 (in XX cells) or 24 (in XY cells); all linear
one circular DNA molecule
Total number of DNA molecules per cell
varies according to ploidy; 46 in diploid cells
often several thousand copies (but copy number
varies in diﬀerent cell types)
Associated protein
several classes of histone and nonhistone protein
largely free of protein
Number of protein-coding genes
~21,000
13
Number of RNA genes
uncertain, but >6000
24
Gene density
~1/120 kb, but great uncertainty
1/0.45 kb
Repetitive DNA
more than 50% of genome; see Figure 9.1
very little
Transcription
genes are often independently transcribed
multigenic transcripts are produced from both
the heavy and light strands
Introns
found in most genes
absent
Percentage of protein-coding DNA
~1.1%
~66%
Codon usage
61 amino acid codons plus three stop codonsa
60 amino acid codons plus four stop codonsa
Recombination
at least once for each pair of homologs at meiosis
not evident
Inheritance
Mendelian for X chromosome and autosomes;
paternal for Y chromosome
exclusively maternal
aFor details see Figure 1.25.
In addition to their differences in genetic capacity and different genetic codes,
the mitochondrial and nuclear genomes differ in many other aspects of their
organization and expression (Table 9.2).
The human nuclear genome consists of 24 widely diﬀerent
chromosomal DNA molecules
The human nuclear genome is 3.1 Gb (3100 Mb) in size. It is distributed between
24 different types of linear double-stranded DNA molecule, each of which has
histones and nonhistone proteins bound to it, constituting a chromosome. There
are 22 types of autosome and two sex chromosomes, X and Y. Human chromosomes can easily be differentiated by chromosome banding (see Figure 2.15), and
have been classiﬁed into groups largely according to size and, to some extent,
centromere position (see Table 2.3).
There is a single nuclear genome in sperm and egg cells and just two copies in
most somatic cells, in contrast to the hundreds or even thousands of copies of the
mitochondrial genome. Because the size of the nuclear genome is about 186,000
times the size of a mtDNA molecule, however, the nucleus of a human cell typically contains more than 99% of the DNA in the cell; the oocyte is a notable exception because it contains as many as 100,000 mtDNA molecules.
Not all of the human nuclear genome has been sequenced. The Human
Genome Project focused primarily on sequencing euchromatin, the gene-rich,
transcriptionally active regions of the nuclear genome that account for 2.9 Gb.
The other 200 Mb is made up of permanently condensed and transcriptionally
inactive (constitutive) heterochromatin. The heterochromatin is composed of
long arrays of highly repetitive DNA that are very difﬁcult to sequence accurately.
For a similar reason, the long arrays of tandemly repeated transcription units
encoding 28S, 18S, and 5.8S rRNA were also not sequenced.
The DNA of human chromosomes varies considerably in length and also in
the proportions of underlying euchromatin and constitutive heterochromatin
(Table 9.3). Each chromosome has some constitutive heterochromatin at the
GENERAL ORGANIZATION OF THE HUMAN GENOME
261
TABLE 9.3 DNA CONTENT OF HUMAN CHROMOSOMES
Chromosome
Total DNA
(Mb)
Euchromatin
(Mb)
Heterochromatin
(Mb)
Chromosome
Total DNA
(Mb)
Euchromatin
(Mb)
Heterochromatin
(Mb)
1
249
224
19.5
13
115
96.3
17.2
2
243
240
2.9
14
107
88.3
17.2
3
198
197
1.5
15
103
82.1
18.3
4
191
188
3.0
16
90
79.0
10.0
5
181
178
0.3
17
81
78.7
7.5
6
171
168
2.3
18
78
74.6
1.4
7
159
156
4.6
19
59
60.8
0.3
8
146
143
2.2
20
63
60.6
1.8
9
141
120
18.0
21
48
34.2
11.6
10
136
133
2.5
22
51
35.1
14.3
11
135
131
4.8
X
155
151
3.0
12
134
131
4.3
Y
59
26.4
31.6
Chromosome sizes are taken from the ENSEMBL Human Map View (http://www.ensembl.org/Homo_sapiens/Location/Genome). Heterochromatin
ﬁgures are estimates abstracted from International Human Genome Sequencing Consortium (2004) Nature 431, 931–945. The size of the total human
genome is estimated to be about 3.1 Gb, with euchromatin accounting for close to 2.9 Gb and heterochromatin accounting for 200 Mb.
centromere. Certain chromosomes, notably 1, 9, 16, and 19, also have signiﬁcant
amounts of heterochromatin in the euchromatic region close to the centromere
(pericentromere), and the acrocentric chromosomes each have two sizeable heterochromatic regions. But the most signiﬁcant representation is in the Y chromosome, where most of the DNA is organized as heterochromatin.
The base composition of the euchromatic component of the human genome
averages out at 41% (G+C), but there is considerable variation between chromosomes, from 38% (G+C) for chromosomes 4 and 13 up to 49% (for chromosome
19). It also varies considerably along the lengths of chromosomes. For example,
the average (G+C) content on chromosome 17q is 50% for the distal 10.3 Mb but
drops to 38% for the adjacent 3.9 Mb. There are regions of less than 300 kb with
even wider swings, for example from 33.1% to 59.3% (G+C).
The proportion of some combinations of nucleotides can vary considerably.
Like other vertebrate nuclear genomes, the human nuclear genome has a conspicuous shortage of the dinucleotide CpG. However, certain small regions of
transcriptionally active DNA have the expected CpG density and, signiﬁcantly,
are unmethylated or hypomethylated (CpG islands; Box 9.1).
The human genome contains at least 26,000 genes, but the exact
gene number is diﬃcult to determine
Several years after the Human Genome Project delivered the ﬁrst reference
genome sequence, there is still very considerable uncertainty about the total
human gene number. When the early analyses of the genome were reported in
2001, the gene catalog generated by the International Human Genome Sequencing Consortium was very much oriented toward protein-coding genes. Original
estimates suggested more than 30,000 human protein-coding genes, most of
which were gene predictions without any supportive experimental evidence. This
number was an overestimate because of errors that were made in deﬁning genes
(see Box 8.5).
To validate gene predictions supportive evidence was sought, mostly by evolutionary comparisons. Comparison with other mammalian genomes, such as
262
Chapter 9: Organization of the Human Genome
BOX 9.1 ANIMAL DNA METHYLATION AND VERTEBRATE CpG ISLANDS
DNA methylation in multicellular animals often involves methylation
of a proportion of cytosine residues, giving 5-methylcytosine (mC).
In most animals (but not Drosophila melanogaster), the dinucleotide
CpG is a common target for cytosine methylation by speciﬁc cytosine
methyltransferases, forming mCpG (Figure 1A).
DNA methylation has important consequences for gene
expression and allows particular gene expression patterns to be stably
transmitted to daughter cells. It has also been implicated in systems
of host defense against transposons. Vertebrates have the highest
levels of 5-methylcytosine in the animal kingdom, and methylation
is dispersed throughout vertebrate genomes. However, only a small
percentage of cytosines are methylated (about 3% in human DNA,
mostly as mCpG but with a small percentage as mCpNpG, where N is
any nucleotide).
5-Methylcytosine is chemically unstable and is prone to
deamination (see Figure 1A). Other deaminated bases produce
derivatives that are identiﬁed as abnormal and are removed by the
DNA repair machinery (e.g. unmethylated cytosine produces uracil
when deaminated). However, 5-methyl cytosine is deaminated to
give thymine, a natural base in DNA that is not recognized as being
abnormal by cellular DNA repair systems. Over evolutionarily long
periods, therefore, the number of CpG dinucleotides in vertebrate
DNA has gradually fallen because of the slow but steady
conversion of CpG to TpG (and to CpA on the complementary
strand; Figure 1B).
Although the overall frequency of CpG in the vertebrate genome
is low, there are small stretches of unmethylated or hypomethylated
DNA that are characterized by having the normal, expected CpG
frequency. Such islands of normal CpG density (CpG islands) are
comparatively GC-rich (typically more than 50% GC) and extend over
hundreds of nucleotides. CpG islands are gene markers because they
are associated with transcriptionally active regions. Highly methylated
DNA regions are prone to adopting a condensed chromatin
conformation, but for actively transcribing DNA the chromatin needs
to be in a more extended, open unmethylated conformation that
allows various regulatory proteins to bind more readily to promoters
and other gene control regions.
(A)
cytosine
(B)
NH2
4C
m
CpG
m
GpC
3¢
5¢
CH
deamination
CH
C
O
3¢
5
3N
2
5¢
NH
6
1
methylation
at 5¢ carbon
5¢
3¢
TpG
m
GpC
NH2
3¢
5¢
DNA duplication
C
N
C
C
CH
CH3
NH
O
5¢
TpG
3¢
3¢
ApC
5¢
5¢
CpG
m
GpC
+
5-methylcytosine
deamination
3¢
3¢
5¢
O
C
C
HN
C
O
CH3
CH
NH
thymine
(forms mismatch with G;
inefficiently recognized by
DNA repair system)
Figure 1 Instability of vertebrate CpG dinucleotides. (A) The cytosine
in CpG dinucleotides is a target for methylation at the 5¢ carbon atom.
The resulting 5-methylcytosine is deaminated to give thymine (T), which
is ineﬃciently recognized by the DNA repair system and so tends to
persist (however, deamination of unmethylated cytosine gives uracil
which is readily recognized by the DNA repair system). (B) The vertebrate
CpG dinucleotide is gradually being replaced by TpG and CpA.
those of the mouse and the dog, failed to identify counterparts of many of the
originally predicted human genes. By late 2009 the estimated number of human
protein-coding genes appeared to be stabilizing somewhere around 20,000 to
21,000, but huge uncertainty remained about the number of human RNA genes.
RNA genes are difﬁcult to identify by using computer programs to analyze
genome sequences: there are no open reading frames to screen for, and many
RNA genes are very small and often not well conserved during evolution. There is
also the problem of how to deﬁne an RNA gene. As we detail in Chapter 12, comprehensive analyses have recently suggested that the great majority of the
genome—and probably at least 85% of nucleotides—is transcribed. It is currently
unknown how much of the transcriptional activity is background noise and how
much is functionally signiﬁcant.
By mid-2009, evidence for at least 6000 human RNA genes had been obtained,
including thousands of genes encoding long noncoding RNAs that are thought to
be important in gene regulation. In addition, there is evidence for tens of thousands of different tiny human RNAs, but in many such cases quite large numbers
of different tiny RNAs are obtained by the processing of single RNA transcripts.
We look at noncoding RNAs in detail in Section 9.3.
The combination of about 20,000 protein-coding genes and at least 6000 RNA
genes gives a total of at least 26,000 human genes. This remains a provisional
total gene number; deﬁning RNA genes is challenging and it will be some time
before we obtain an accurate human gene number.
GENERAL ORGANIZATION OF THE HUMAN GENOME
Human genes are unevenly distributed between and within
chromosomes
Human genes are unevenly distributed on the nuclear DNA molecules. The constitutive heterochromatin regions are devoid of genes and, even within the
euchromatic portion of the genome, gene density can vary substantially between
chromosomal regions and also between whole chromosomes.
The ﬁrst general insight into how genes are distributed across the human
genome was obtained when puriﬁed CpG island fractions were hybridized to
metaphase chromosomes. CpG islands have long been known to be strongly
associated with genes (see Box 9.1). On this basis, it was concluded that gene
density must be high in subtelomeric regions, and that some chromosomes
(e.g. 19 and 22) are gene-rich whereas others (e.g. X and 18) are gene-poor (see
Figure 8.17). The predictions of differential CpG island density and differential
gene density were subsequently conﬁrmed by analyzing the human genome
sequence.
This difference in gene density can also be seen with Giemsa staining (G banding) of chromosomes. Regions with a low (G+C) content correlate with the darkest G bands, and those with a high (G+C) content with pale bands. GC-rich chromosomes (e.g. chromosome 19) and regions (e.g. pale G bands) are also
comparatively rich in genes. For example, the gene-rich human leukocyte antigen (HLA) complex (180 protein-coding genes in a span of 4 Mb) is located within
the pale 6p21.3 band. In striking contrast, the mammoth dystrophin gene extends
over 2.4 Mb of DNA in a dark G band at Xp21.2 without evidence for any other
protein-coding gene in this region.
Duplication of DNA segments has resulted in copy-number
variation and gene families
Small genomes, such as those of bacteria and mitochondria, are typically tightly
packed with genetic information that is presented in extremely economical
forms. Large genomes, such as the nuclear genomes of eukaryotes, and especially vertebrate genomes, have the luxury of not being so constrained. Repetitive
DNA is one striking feature of large genomes, in both abundance and
importance.
Different types of DNA sequence can be repeated. Some are short noncoding
sequences that are present in a few copies to millions of copies. These will be
discussed further in Section 9.4. Many others are moderately long to large DNA
sequences that often contain genes or parts of genes. Such duplicated sequences
are prone to various genetic mechanisms that result in copy-number variation
(CNV) in which the number of copies of speciﬁc moderately long sequences—
often from many kilobases to several megabases long—varies between different
haplotypes. Copy-number variation generates a type of structural variation that
we consider more fully in Chapter 13, but we will consider some of the mechanisms below in the context of how genes become duplicated. It is clear, however,
that CNV is quite extensive in the human genome. For example, when James
Watson’s genome was sequenced, 1.4% of the total sequencing data obtained did
not map with the reference human genome sequence. As personal genome
sequencing accelerates, new CNV regions are being identiﬁed with important
implications for gene expression and disease.
Repeated duplication of a gene-containing sequence gives rise to a gene family. As we will see in Sections 9.2 and 9.3, many human genes are members of
multigene families that can vary enormously in terms of copy number and distribution. They arise by one or more of a variety of different mechanisms that result
in gene duplication. Gene families may also contain evolutionarily related
sequences that may no longer function as working genes (pseudogenes).
Gene duplication mechanisms
Gene duplication has been a common event in the evolution of the large nuclear
genomes found in complex eukaryotes. The resulting multigene families have
from two to very many gene copies. The gene copies may be clustered together in
one subchromosomal location or they may be dispersed over several chromosomal locations. Several different types of gene duplication can occur:
263
264
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Chapter 9: Organization of the Human Genome
Tandem gene duplication typically arises by crossover between unequally
aligned chromatids, either on homologous chromosomes (unequal crossover)
or on the same chromosome (unequal sister chromatid exchange). Figure 9.5
shows the general mechanism. The repeated segment may be just a few kilobases long or may be quite large and contain from one to several genes. Two
such repeats are said to be direct repeats if the head of one repeat joins the tail
of its neighbor (ÆÆ) or inverted repeats if there is joining of heads (Æ¨) or
tails (¨Æ). Over a long (evolutionary) time-scale the duplicated sequences
can be separated on the same chromosome (by a DNA insertion or inversion)
or become distributed on different chromosomes by translocations.
•
Duplicative transposition describes the process by which a duplicated DNA
copy integrates into a new subchromosomal location. Typically this involves
retrotransposition: cellular reverse transcriptases make a cDNA copy of an
RNA transcript, whereupon the cDNA copy integrates into a new chromosomal location. The same type of mechanism can often lead to defective gene
copies and will be detailed in Section 9.2.
•
Gene duplication by ancestral cell fusion. Aerobic eukaryotic cells are thought
to have evolved through the endocytosis of a type of bacterial cell by a eukaryotic precursor cell. The current mitochondrial genome is thought to have
derived from the bacterial cell’s genome but is now a very small remnant
because many of the original bacterial genes were subsequently excised and
transferred to what is now the nuclear genome. As a result, the nuclear genome
contains duplicated genes that encode cytoplasm-speciﬁc and mitochondrion-speciﬁc isoforms for certain enzymes and certain other key proteins.
•
Large-scale subgenomic duplications can arise as a result of chromosome
translocations. Euchromatic regions close to human centromeres and to
telomeres (pericentromeric and subtelomeric regions, respectively) are comparatively unstable and are prone to recombination with other chromosomes.
As a result, large segments of DNA containing multiple genes have been duplicated. Within the past 40 million years of primate evolution, this process has
led to the duplication of about 400 large (several megabases long) DNA segments, accounting for more than 5% of the euchromatic genome. This type of
duplication, known as segmental duplication, results in very high (often
more than 95%) sequence identity between the DNA copies and can involve
both intrachromosomal duplications and also interchromosomal duplications (Figure 9.6). Segmental duplications are important contributors to
copy-number variation and to chromosomal rearrangements leading
to disease and rapid gene innovation. We consider how they originate in
Chapter 10.
•
Whole genome duplication. It is now clear from comparative genomics studies that whole genome duplication has occurred at several times during evolution on a variety of different eukaryotic lineages. For example, there is compelling evidence that whole genome duplication occurred in the early
evolution of chordates. This type of event could explain why vertebrates have
four HOX clusters (see Figure 5.5). Whole genome duplication is detailed in
Chapter 10 in the context of genome evolution.
1
2
3
4
5
6
7
16
10
11
14
15
17 18 19 20
22
A
A
A
A
+
Figure 9.5 Tandem gene duplication.
Crossover between misaligned chromatids
can result in one chromosome with
a tandem duplication of a sequence
containing a gene (such as gene A shown
here by a green box) and one in which the
gene is lost. The mispairing of chromatids
may be stabilized by closely related
members of an interspersed repeated DNA
family such as Alu repeats (as shown here by
orange boxes). The crossover event leading
to tandem gene duplication may result
from unequal crossover (crossover between
misaligned chromatids on homologous
chromosomes) or unequal sister chromatid
exchange (the analogous process by which
sister chromatids are misaligned; see Figure
13.3 for an illustration).
Figure 9.6 Segmental duplication. The
horizontal bar in the center is a linear
map of the DNA of human chromosome
16 (the central green segment represents
heterochromatin). The black horizontal bars
at the top and bottom represent linear maps
of 16 other chromosomes containing large
segments that are shared with chromosome
16, with red connecting lines marking
the positions of homologous sequences.
Intrachromosomal duplications are shown
by blue chevrons (^) linking the positions of
large duplicated sequences on chromosome
16. [From Martin J, Han C, Gordon LA
et al. (2004) Nature 432, 988–994. With
permission from Macmillan Publishers Ltd.
For chromosomal coordinates and further
information on segmental duplications in
the human genome, dedicated segmental
duplication databases can be accessed at
http://humanparalogy.gs.washington.edu/
and http://projects.tcag.ca/humandup/]
PROTEIN-CODING GENES
9.2
265
PROTEINCODING GENES
For many years, molecular geneticists believed that the major functional endpoint of DNA was protein. Studies of prokaryotic genomes supported this belief,
partly because these genomes are rich in protein-coding DNA. It came as a surprise to ﬁnd that the much larger genomes of complex eukaryotes have comparatively little protein-coding DNA. For example, protein-coding DNA sequences
account for close to 90% of the E. coli genome but just 1.1% of the human
genome.
Human protein-coding genes show enormous variation in size
and internal organization
Size variation
Genes in simple organisms such as bacteria are comparatively similar in size and
are usually very short (typically about 1 kb long). In complex eukaryotes, genes
can show huge variation in size. Although there is generally a direct correlation
between gene and product sizes, there are some striking anomalies. For example,
the giant 2.4 Mb dystrophin gene is more than 50 times the size of the apolipoprotein B gene but the dystrophin protein has a linear length (total amino acid
number) that is about 80% of that of apolipoprotein B (Table 9.4).
A small minority of human protein-coding genes lack introns and are generally small (see note to Table 9.4 for some examples). For those that do possess
TABLE 9.4 STRUCTURAL VARIATION IN SIZE AND ORGANIZATION OF HUMAN PROTEINCODING GENES
Human protein
Size of protein (no.
of amino acids)
Size of gene
(kb)
No. of
exons
Coding DNA (%)
Average size of
exon (bp)
Average size of
intron (bp)
SRY
204
0.9
1
94
850
–
b-Globin
146
1.6
3
38
150
490
p16
156
7.4
3
17
406
3064
Serum albumin
609
18
14
12
137
1100
Type VII collagen
2928
31
118
29
77
190
393
39
10
6.0
236
3076
Complement C3
1641
41
29
8.6
122
900
Apolipoprotein B
4563
45
29
31
487
1103
452
90
26
3
96
3500
Factor VIII
2351
186
26
3
375
7100
Huntingtin
3144
189
67
8.0
201
2361
928
198
27
2.4
179
6668
1480
250
27
2.4
227
9100
34,350
283
363
40
315
466
Utrophin
3433
567
74
2.2
168
7464
Dystrophin
3685
2400
79
0.6
180
30,770
p53
Phenylalanine hydroxylase
RB1 retinoblastoma protein
CFTR (cystic ﬁbrosis
transmembrane receptor)
Titin
Where isoforms are evident, the given ﬁgures represent the largest isoforms. As genes get larger, exon size remains fairly constant but intron sizes can
become very large. Internal exons tend to be fairly uniform in size, but the terminal exon or some exons near the 3¢ end can be many kilobases long;
for example, exon 26 of the APOB gene is 7.5 kb long. Note the extraordinarily high exon content and comparatively small intron sizes in the genes
encoding type VII collagen and titin. In addition to SRY, other single-exon protein-coding genes in the nuclear genome include retrogenes (see
Table 9.8) and genes encoding other SOX proteins, interferons, histones, many G-protein-coupled receptors, heat shock proteins, many ribonucleases,
and various neurotransmitter receptors and hormone receptors.
266
Chapter 9: Organization of the Human Genome
introns, there is an inverse correlation between gene size and fraction of coding
DNA (see Table 9.4). This does not arise because exons in large genes are smaller
than those in small genes. The average exon size in human genes is close to
300 bp, and exon size is comparatively independent of gene length. Instead, there
is huge variation in intron lengths, and large genes tend to have very large introns
(see Table 9.4). Transcription of long introns is, however, costly in time and energy;
transcription of the 2.4 Mb dystrophin gene takes about 16 hours. Thus, very
highly expressed genes often have short introns or no introns at all.
Repetitive sequences within coding DNA
Highly repetitive DNA sequences are often found within introns and ﬂanking
sequences of genes. They will be detailed in Section 9.4. In addition, repetitive
DNA sequences are found to different extents in exons. Tandem repetition of very
short oligonucleotide sequences (1–4 bp) is frequent and may simply reﬂect statistically expected frequencies for certain base compositions. Tandem repetition
of sequences encoding known or assumed protein domains is also quite common, and it may be functionally advantageous by providing a more available biological target.
The sequence identities between the repeated protein domains are often
quite low but can sometimes be high. Lipoprotein Lp (a), encoded by the LPA
gene on chromosome 6q26, provides a classical example. It contains multiple
tandemly repeated kringle domains, which are each about 114 amino acids long
and form disulﬁde-bonded loops. The different kringle domains are often nearly
identical in amino acid sequence. Even at the nucleotide sequence level the DNA
repeats that encode the kringle domains show very high levels of sequence identity, making them prone to unequal crossover. As a result, the LPA gene is subject
to length polymorphism, and the number of kringle domains in lipoprotein Lp
(a) varies but is usually 15 or more.
Diﬀerent proteins can be speciﬁed by overlapping transcription
units
Overlapping genes and genes-within-genes
Simple genomes have high gene densities (roughly one per 0.5, 1, and 2 kb for the
genomes of human mitochondria, Escherichia coli, and Saccharomyces cerevisiae, respectively) and often show examples of partly overlapping genes. Different
reading frames may be used, sometimes from the same sense strand. In complex
organisms, such as humans, genes are much bigger, and there is less clustering of
protein-coding sequences (Table 9.5).
Gene density varies enormously from chromosome to chromosome and
within different regions of the same chromosome. In chromosomal regions with
high gene density, overlapping genes may be found; they are typically transcribed
from opposing DNA strands. For example, the class III region of the HLA complex
at 6p21.3 has an average gene density of about one gene per 15 kb and is known
to contain several examples of partly overlapping genes (Figure 9.7A).
Whole genome analyses show that about 9% of human protein-coding genes
overlap another such gene. More than 90% of the overlaps involve genes transcribed from opposing strands. Sometimes the overlaps are partial, but in other
cases small protein-coding genes are located within the introns of larger genes.
The NF1 (neuroﬁbromatosis type I) gene, for example, has three small internal
genes transcribed from the opposite strand (Figure 9.7B).
Recent analyses have also shown that RNA genes can frequently overlap protein-coding genes. The positioning of RNA genes will be covered in Section 9.3.
Genes divergently transcribed or co-transcribed from a common
promoter
Some protein-coding genes share a promoter. In many cases the 5¢ ends of the two
genes are often separated by just a few hundred nucleotides and the genes are
transcribed in opposite directions from the common promoter. This type of bidirectional gene organization may provide for common regulation of the gene pair.
Alternatively, genes with a common promoter are transcribed in the same
direction to produce multigenic transcripts that are then cleaved to produce a
PROTEIN-CODING GENES
TABLE 9.5 HUMAN GENOME AND HUMAN GENE STATISTICS
SIZE OF GENOME COMPONENTS
Mitochondrial genome
16.6 kb
Nuclear genome
3.1 Gba
Euchromatic component
2.9 Gb (~93%)
Highly conserved fraction
~150 Mb (~5%)
Protein-coding DNA sequences
~35 Mb (~1.1%)
Other highly conserved DNA
~115 Mb (~3.9%)
Segmentally duplicated DNA
~160 Mb (~5.5%)
Highly repetitive DNA
~1.6 Gb (~50%)
Constitutive heterochromatin
~ 200 Mb (~7%; Table 9.3)
Transposon-based repeats
~1.4 Gb (~45%; Table 9.12)
DNA per chromosome
48 Mb—249 Mb (Table 9.3)
GENE NUMBER
Nuclear genome
> 26,000
Mitochondrial genome
37
Protein-coding genes
~ 20,000–21,000
RNA genes
> 6000 (exact ﬁgure not known)
Pseudogenes related to protein-coding genes
> 12,000
GENE DENSITY
Nuclear genome
>1 per 120 kb (but considerable uncertainty)
Mitochondrial genome
1 per 0.45 kb
LENGTH OF PROTEINCODING GENES
Average length
53.6 kb
Smallest
a few hundred base pairs long (several examples)
Largest
2.4 Mb (dystrophin)
EXON NUMBER IN PROTEINCODING GENES
Average number of exons in one geneb
9.8
Largest number in one gene
363 (in the titin gene)
Smallest number in one gene
1 (no introns—see Tables 9.4 and 9.7 for example)
EXON SIZE IN PROTEINCODING GENES
Average exon size
288 bp (but exons at 3¢ end of genes tend to be
large)
Smallest
< 10 bp (various; e.g. exon 3 of the troponin I gene
TNNI1 is just 4 bp long)
Largest
18.2 kb (exon 6 in MUC16 isoform-201)
INTRON SIZE IN PROTEINCODING GENES
Smallest
< 30 bp (various)
Largest
1.1 Mb (intron 5 in KCNIP4)
RNA SIZE
Smallest noncoding RNA
< 20 nucleotides (e.g. many transcriptional start
site-associated RNAs are 18 nucleotides)
Largest noncoding RNA
Several hundred thousand nucleotides; e.g. UBE3A
antisense RNA is likely to be close to 1 Mb
Largest mRNA
> 103 kb (titin mRNA, NF-2A isoform)
POLYPEPTIDE SIZE
Smallest
tens of amino acids (various neuropeptides)
Largest
34,350 amino acids (titin, NF-2A isoform)
aNote that the total size can vary between haplotypes because of copy-number variation. bFor the
longest isoform. Data were obtained largely from ENSEMBL release 55 datasets.
267
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Chapter 9: Organization of the Human Genome
(A)
0
900 kb
LTB
1C7
CYP C4B CYP C4A
G5b CKIIB
NOB1
21B
21Ps G11
B144
NB6
TNF
G16
Hsp70 G7c BAT5 G5 G3a
NOB2
1kBL MICA
YB ZB YA ZA G11a
LTA
RAGE
PERB10
HOM G7d
RD G10
XA
P5-6
G4 K18L
G15 TN-X
PBX2
Bf
G6
G9 2 G7b
DHFRPs
BAT1 PERB6
G3 G1
G14
XB-S
G18
17 NOB3
G2
C2 C9a G8
G7a G7 G8
NOTCH4
MICB
PPIPs
(B)
exon 27b
exon 28
intron 27b
sense strand
5¢
of NF1 gene
antisense strand
3¢
of NF1 gene
3¢
5¢
OGMP
EVI2B
EVI2A
2.2 kb
10 kb
4 kb
Figure 9.7 Overlapping genes and genes-within-genes. (A) Genes in the class III region of the HLA complex are tightly packed and
overlapping in some cases. Arrows show the direction of transcription. (B) Intron 27b of the NF1 (neuroﬁbromatosis type I) gene is 60.5 kb
long and contains three small internal genes, each with two exons, which are transcribed from the opposing strand. The internal genes (not
drawn to scale) are OGMP (oligodendrocyte myelin glycoprotein) and EVI2A and EVI2B (human homologs of murine genes thought to be
involved in leukemogenesis and located at ecotropic viral integration sites).
separate transcript for each gene. Such genes are said to form part of a polycistronic (= multigenic) transcription unit. Polycistronic transcription units are
common in simple genomes such as those of bacteria and the mitochondrial
genome (see Figure 9.3). Within the nuclear genome, some examples are known
of different proteins being produced from a common transcription unit. Typically,
they are produced by cleavage of a hybrid precursor protein that is translated
from a common transcript. The A and B chains of insulin, which are intimately
related functionally, are produced in this way (see Figure 1.26), as are the related
peptide hormones somatostatin and neuronostatin. Sometimes, however, functionally distinct proteins are produced from a common protein precursor. The
UBA52 and UBA80 genes, for example, both generate ubiquitin and an unrelated
ribosomal protein (S27a and L40, respectively).
More recent analyses have shown that the long-standing idea that most
human genes are independent transcription units is not true, and so the deﬁnition of a gene will need to be radically revised. Multigenic transcription is now
known to be rather frequent in the human genome, and speciﬁc proteins and
functional noncoding RNAs can be made by common RNA precursors. This will
be explored further in Section 9.3.
Human protein-coding genes often belong to families of genes
that may be clustered or dispersed on multiple chromosomes
Duplicated genes and duplicated coding sequence components are a common
feature of animal genomes, especially large vertebrate genomes. As we will see in
Chapter 10, gene duplication has been an important driver in the evolution of
functional complexity and the origin of increasingly complex organisms. Genes
that operate in the same or similar functional pathways but produce proteins
with little evidence of sequence similarity are distantly related in evolution, and
they tend to be dispersed at different chromosomal locations. Examples include
genes encoding insulin (on chromosome 11p) and the insulin receptor (19p); ferritin heavy chain (11q) and ferritin light chain (22q); steroid 11-hydroxylase (8q)
and steroid 21-hydroxylase (6p); and JAK1 (1p) and STAT1 (2q). However, genes
that produce proteins with both structural and functional similarity are often
organized in gene clusters.
PROTEIN-CODING GENES
0
10
20
30
40
50
yx1 ya2 ya1 a2
x2
a1
Figure 9.8 Examples of human clustered
gene families. Genes in a cluster are often
closely related in sequence and are typically
transcribed from the same strand. Gene
clusters often contain a mixture of expressed
genes and nonfunctional pseudogenes. The
functional status of the q-globin and CS-L
genes is uncertain. The scales at the top
(globin and growth hormone clusters) and
the bottom (albumin cluster) are in kilobases.
60
q
a-globin cluster 16p13
e
Gg Ag
yb
d
b
b-globin cluster
11p15
growth hormone
cluster 17q23
hGH-N CS-L
albumin
cluster 4q12
ALB
0
AFP
20
40
expressed gene
CS-A
hGH-V
CS-B
ALF
60
80
GC/DBP
100
120
140
expressed, but status unknown
269
160
180
pseudogene
Different classes of human gene families can be recognized according to the
degree of sequence similarity and structural similarity of their protein products.
If two different genes make very similar protein products, they are most likely to
have originated by an evolutionarily very recent gene duplication, most probably
some kind of tandem gene duplication event, and they tend to be clustered
together at a speciﬁc subchromosomal location. If they make proteins that are
more distantly related in sequence, they most probably arose by a more ancient
gene duplication. They may originally have been clustered together, but over
long evolutionary time-scales the genes could have been separated by translocations or inversions, and they tend to be located at different chromosomal
locations.
Some gene families are organized in multiple clusters. The b-, g-, d-, and
e-globin genes are located in a gene cluster on 11p and are more closely related to
each other than they are to the genes in the a-globin gene cluster on 16p (Figure
9.8). The genes in the b-globin gene cluster on 11p originated by gene duplication events that were much more recent in evolution than the early gene
duplication event that gave rise to ancestors of the a- and b-globin genes. An
outstanding example of a gene family organized as multiple gene clusters is the
olfactory receptor gene family. The genes encode a diverse repertoire of receptors
that allow us to discriminate thousands of different odors; the genes are located
in large clusters at multiple different chromosomal locations (Table 9.6).
Some gene families have individual gene copies at two or more chromosomal
locations without gene clustering (see Table 9.6). The genes at the different locations are usually quite divergent in sequence unless gene duplication occurred
relatively recently or there has been considerable selection pressure to maintain
sequence conservation. The family members are expected to have originated
from ancient gene duplications.
Diﬀerent classes of gene family can be recognized according to
the extent of sequence and structural similarity of the protein
products
As listed below, various classes of gene family can be distinguished according to
the level of sequence identity between the individual gene members.
• In gene families with closely related members, the genes have a high degree of
sequence homology over most of the length of the gene or coding sequence.
Examples include histone gene families (histones are strongly conserved, and
subfamily members are virtually identical), and the a-globin and b-globin
gene families.
270
Chapter 9: Organization of the Human Genome
TABLE 9.6 EXAMPLES OF CLUSTERED AND INTERSPERSED MULTIGENE FAMILIES
Family
Copy no.
Organization
Chromosome location(s)
Growth hormone gene cluster
5
clustered within 67 kb; one pseudogene (Figure 9.8)
17q24
a-Globin gene cluster
7
clustered over ~50 kb (Figure 9.8)
16p13
Class I HLA heavy chain genes
~20
clustered over 2 Mb (Figure 9.10)
6p21
HOX genes
38
organized in four clusters (Figure 5.5)
2q31, 7p15, 12q13, 17q21
Histone gene family
61
modest-sized clusters at a few locations; two large clusters
on chromosome 6
many
Olfactory receptor gene family
> 900
about 25 large clusters scattered throughout the genome
many
Aldolase
5
three functional genes and two pseudogenes on ﬁve
diﬀerent chromosomes
many
PAX
9
all nine are functional genes
many
NF1 (neuroﬁbromatosis type I)
> 12
one functional gene at 22q11; others are nonprocessed
pseudogenes or gene fragments (Figure 9.11)
many, mostly pericentromeric
Ferritin heavy chain
20
one functional gene on chromosome 11; most are
processed pseudogenes
many
CLUSTERED GENE FAMILIES
INTERSPERSED GENE FAMILIES
•
In gene families deﬁned by a common protein domain, the members may
have very low sequence homology but they possess certain sequences that
specify one or more speciﬁc protein domains. Examples include the PAX gene
family and SOX gene family (Table 9.7).
•
Examples of gene families deﬁned by functionally similar short protein motifs
are families of genes that encode functionally related proteins with a DEAD
box motif (Asp-Glu-Ala-Asp) or the WD repeat (Figure 9.9).
Some genes encode products that are functionally related in a general sense
but show only very weak sequence homology over a large segment, without very
signiﬁcant conserved amino acid motifs. Nevertheless, there may be some evidence for common general structural features. Such genes can be grouped into
an evolutionarily ancient gene superfamily with very many gene members.
Because multiple different gene duplication events have occurred periodically
during the long evolution of a gene superfamily, some of the gene members make
proteins that are very divergent in sequence from those of some other family
members, but genes resulting from more recent duplications are more readily
seen to be related in sequence.
TABLE 9.7 EXAMPLES OF HUMAN GENES WITH SEQUENCE MOTIFS THAT ENCODE HIGHLY CONSERVED DOMAINS
Gene family
Number of genes
Sequence motif/domain
Homeobox genes
38 HOX genes plus 197
orphan homeobox genes
homeobox speciﬁes a homeodomain of ~60 amino acids; a wide variety of diﬀerent
subclasses have been deﬁned
PAX genes
9
paired box encodes a paired domain of ~124 amino acids; PAX genes often also have a
type of homeodomain known as a paired-type homeodomain
SOX genes
19
SRY-like HMG box which encodes a domain of 70–80 amino acids
TBX genes
14
T-Box encodes a domain of ~170 amino acids
Forkhead domain genes
50
the forkhead domain is ~110 amino acids long
POU domain genes
16
the POU domain is ~150 amino acids long
PROTEIN-CODING GENES
(A) DEAD box
NH2
22–42
19–29 17–29
17–23
19–51 115–192
20–25
DEAD
AxxGxGKT
PTRELA
GG
TPGR
SAT
ARGxD
HRIGR
(B)
6–94
WD repeat
COOH
23–41
GH
WD
n = 4–14
core
Two important examples of gene superfamilies are the Ig (immunoglobulin)
and GPCR (G-protein-coupled receptor) superfamilies. Members of the Ig superfamily all have globular domains resembling those found in immunoglobulins,
and in addition to immunoglobulins they include a variety of cell surface proteins and soluble proteins involved in the recognition, binding, or adhesion processes of cells (see Figure 4.22 for some examples). The GPCR superfamily is very
large, with at least 799 unique full-length members distributed throughout the
human genome. All the GPCR proteins have a common structure of seven a-helix
transmembrane segments, but they typically have low (less than 40%) sequence
similarity to each other. They mediate ligand-induced cell signaling via interaction with intracellular G proteins, and most work as rhodopsin receptors.
Gene duplication events that give rise to multigene families also
create pseudogenes and gene fragments
Gene families frequently have defective gene copies in addition to functional
genes. A defective gene copy that contains at least multiple exons of a functional gene is known as a pseudogene (Box 9.2). Other defective gene copies may
have only limited parts of the gene sequence, sometimes a single exon, and so are
sometimes described as gene fragments.
Clustered gene families often have defective gene copies that have arisen by
tandem duplication. These are examples of nonprocessed pseudogenes. Copying
can be seen to have been performed at the level of genomic DNA because nonprocessed pseudogenes contain counterparts of both exons and introns and
sometimes also of upstream promoter regions. However, even if the copy has
sequences that correspond to the full length of the functional gene, closer examination will identify inappropriate termination codons in exons, aberrant splice
junctions, and so on. Classical examples of nonprocessed pseudogenes are found
in the a-globin and b-globin gene clusters (see Figure 9.8). Sometimes, smaller
truncated gene copies and gene fragment copies are also evident, as in the class I
HLA gene family (Figure 9.10).
A few gene families that are distributed at different chromosomal locations
can also have nonprocessed pseudogene copies of a single functional gene.
Certain types of subchromosomal region, notably pericentromeric and subtelomeric regions, are comparatively unstable. They are prone to recombination
events that can result in duplicated gene segments (containing both exons and
introns) being distributed to other chromosomal locations. The gene copies are
typically defective because they lack some of the functional gene sequence. Two
illustrative examples are sequences related to the NF1 (neuroﬁbromatosis type I)
and the PKD1 (adult polycystic kidney disease) genes.
The NF1 gene is located at 17q11.2. Because of pericentromeric instability,
multiple nonprocessed NF1 pseudogene/gene fragment copies are distributed
over seven different chromosomes, nine being located at pericentromeric regions
(Figure 9.11A). The PKD1 gene has over 46 exons spanning 50 kb and is located
in a subtelomeric region at 16p13.3. Six nonprocessed PKD1 pseudogenes have
been generated by segmental duplications during primate evolution and have
inserted into locations within a region that is about 13–16 Mb proximal to the
PKD1 gene, corresponding to part of band 16p13.1 (Figure 9.11B). The pseudogenes lack sequences at the 3¢ end of the PKD1 gene but have sequence counterparts of much of the genomic sequence spanning exons 1–32, showing 97.6% to
97.8% sequence identity to the PKD1 sequence.
Processed pseudogenes are defective copies of a gene that contain only exonic
sequences and lack an intronic sequence or upstream promoter sequences. They
arise by retrotransposition: cellular reverse transcriptases can use processed gene
271
Figure 9.9 Some gene families encode
functionally related proteins with short
conserved amino acid motifs. (A) DEAD
box family motifs. This gene family encodes
products implicated in cellular processes
involving the alteration of RNA secondary
structure, such as translation initiation and
splicing. Eight very highly conserved amino
acid motifs are evident, including the DEAD
box (Asp-Glu-Ala-Asp). Numbers refer to
frequently found size ranges for intervening
amino acid sequences; X represents any
amino acid. (B) WD repeat family motifs.
This gene family encodes products that
are involved in a variety of regulatory
functions, such as regulation of cell division,
transcription, transmembrane signaling,
and mRNA modiﬁcation. The gene products
are characterized by 4–16 tandem WD
repeats that each contain a core sequence of
ﬁxed length beginning with a GH (Gly-His)
dipeptide and terminating in the dipeptide
WD (Trp-Asp), preceded by a sequence of
variable length.
272
Chapter 9: Organization of the Human Genome
BOX 9.2 THE ORIGINS, PREVALENCE, AND FUNCTIONALITY OF PSEUDOGENES
Pseudogenes are usually thought of as defective copies of a functional
gene to which they show signiﬁcant sequence homology. They
typically arise by some kind of gene duplication event that produces
two gene copies. Selection pressure to conserve gene function need
only be imposed on one gene copy; the other copy can be allowed
to mutate more freely (genetic drift) and can pick up inactivating
mutations, producing a pseudogene. However, some sequences are
referred to as pseudogenes even though they have not originated
by DNA copying. For example, as we will see in Chapter 10, humans
have rare solitary pseudogenes that are clearly orthologs of functional
genes in the great apes and became defective after acquiring harmful
mutations in the human lineage.
Diﬀerent gene duplication mechanisms can give rise to multiple
functional gene copies and defective pseudogenes. Either the
genomic DNA sequence is copied, or a cDNA copy is made (after
reverse transcription of a processed RNA transcript) that integrates
into genomic DNA. For a protein-coding gene, copying at the genomic
DNA level can result in duplication of the promoter and upstream
regulatory sequences as well as of all exons and introns. A defective
gene that derives from a copy of a genomic DNA sequence is known
as a nonprocessed pseudogene (Figure 1A). Such pseudogenes
usually arise by tandem duplication so that they are located close to
functional gene counterparts (see Figures 9.8 and 9.10B for examples),
but some are dispersed as a result of recombination (see Figure 9.11
for examples).
Copying at the cDNA level produces a gene copy that typically
lacks introns, promoter elements, and upstream regulatory
elements. Very occasionally, a processed gene copy can retain some
function (a retrogene; see Table 9.7). However, because they lack
important sequences needed for expression, most processed gene
copies degenerate into processed pseudogenes (sometimes called
retrotransposed pseudogenes; Figure 1B).
Prevalence and functionality of pseudogenes
Eukaryotic genomes typically have many pseudogenes. A longstanding rationale for their abundance is that gene duplication is
evolutionarily advantageous. New functional gene variants can be
created by gene duplication, and pseudogenes have long been viewed
as unsuccessful by-products of the duplication mechanisms. Although
some prokaryotic genomes seem to have many pseudogenes,
pseudogenes are generally rare in prokaryotes because their genomes
are generally designed to be compact.
The great majority of what are conventionally recognized as
human pseudogenes are copies of protein-coding genes simply
because it is relatively easy to identify them (by looking for
frameshifting, splice site mutations, and so on). There are more than
8000 diﬀerent processed pseudogene copies of protein-coding
genes in the human genome, plus more than 4000 nonprocessed
pseudogenes (see the pseudogene database at
http://www.pseudogene.org). Only about 10% of the 21,000 human
protein-coding genes have at least one processed pseudogene, but
highly expressed genes tend to have multiple processed pseudogenes.
For example, the cytoplasmic ribosomal protein contains 95 functional
genes encoding 79 diﬀerent proteins (16 genes are duplicated) and
2090 processed pseudogenes.
RNA pseudogenes are often diﬃcult to identify as pseudogenes
(there is no reading frame to inspect, and RNA genes often lack
introns). Nevertheless, pseudogene copies of many small RNAs are
common (see the table below), notably if they are transcribed by RNA
polymerase III (genes transcribed by RNA polymerase III often have
internal promoters).
RNA family
Number of
human genes
Number of related
pseudogenes
U6 snRNA
49
~800
U7 snRNA
1
85
Y RNA
4
~1000
As will be described in Section 12.4, the Alu repeat, the most
abundant sequence in the human genome, seems to have originated
by the copying of 7SL RNA transcripts, and many other highly
repeated interspersed DNA families in mammals are copies of tRNA.
So, in a sense, RNA pseudogenes have come to be the most common
sequence elements of mammalian genomes.
All the pseudogenes are located in the nuclear genome, but they
do include defective copies of genes that reside in the mitochondrial
genome (mitochondrial pseudogenes). The mitochondrial genome
originated from a much larger bacterial genome and over a long
evolutionary time-scale much of the DNA of the large precursor
mitochondrial genome migrated in a series of independent integration
events into what is now the nuclear genome. mtDNA pseudogenes
now account for at least 0.016% of nuclear DNA (or about 30 times the
content of the mitochondrial genome).
The functionality of pseudogenes has been an enduring debate,
and diﬀerent pseudogene classes have been envisaged. A signiﬁcant
number of pseudogenes (mostly processed pseudogenes) are
transcribed, and antisense pseudogene transcripts may regulate
parent genes. Pseudogenes have also been directly implicated in
the production of endogenous siRNAs that regulate transposons, as
described in Section 9.3. Finally, some pseudogene sequences may be
co-opted for a diﬀerent function. They have been described as exapted
pseudogenes. An example is provided by the XIST gene. It makes a
noncoding RNA that regulates X-chromosome inactivation, and two of
its six exons are known to have originated from a pseudogene copy of
a protein-coding gene.
A
Figure 1 Origins of nonprocessed and processed
pseudogenes. (A) Copying of genomic DNA
sequence containing gene A can produce duplicate
copies of gene A. Strong selection pressure needs
to be applied to one of the copies to maintain
gene function (bold arrow), but the other copy
can be allowed to mutate (dashed arrow). If it
picks up inactivating mutations (red circles), a
nonprocessed pseudogene (yA) can arise.
(B) A processed pseudogene arises after cellular
reverse transcriptases convert a transcript of a gene
into a cDNA that then is able to integrate back into
the genome (see Figure 9.12 for details). The lack
of important sequences such as a promoter usually
results in an inactive gene copy.
A
(A)
(B)
transcription
and processing
gene duplication
mRNA
A
A
mutational
constraint
reverse
transcriptase
cDNA
mutational
flexibility
chromosomal
integration
yA
A
mutation
promoter
inactivating
mutation
non-inactivating
mutation
yA
PROTEIN-CODING GENES
(A)
3¢ UTR
L a1 a2 a3 TM CY
(B)
~2.2 Mb
1
B
C
E
Y
2
Y 3
A
4Y 5Y
G
67
F
L a1 a2
a3
3¢ UTR
3¢ UTR
a2 a3 TM CY
CY
a2 a3 TM CY
3¢ UTR
3¢ UTR
a2
a3 TM CY
transcripts such as mRNA to make cDNA that can then integrate into chromosomal DNA (Figure 9.12). Processed pseudogenes are common in interspersed
gene families (see Table 9.5).
Processed pseudogenes lack a promoter sequence and so are typically not
expressed. Sometimes, however, the cDNA copy integrates into a chromosomal
DNA site that happens, by chance, to be adjacent to a promoter that can drive
expression of the processed gene copy. Selection pressure may ensure that the
processed gene copy continues to make a functional gene product, in which case
it is described as a retrogene. A variety of intronless retrogenes are known to have
testis-speciﬁc expression patterns and are typically autosomal homologs of an
intron-containing X-linked gene (Table 9.8).
One rationale for retrogenes may be a critical requirement to overcome the
lack of expression of certain X-linked sequences in the testis during male meiosis.
During male meiosis, the paired X and Y chromosomes are converted to heterochromatin, forming the highly condensed and transcriptionally inactive XY body.
Autosomal retrogenes can provide the continued synthesis in testis cells of certain crucially important products that are no longer synthesized by genes in the
highly condensed XY body.
(A)
10b
13
27b
NF1
17q11.2
dispersed NF1
pseudogenes
15p11.2 (2 copies)
2q12–q13
14p11<>q11
22p11<>q11
18p11.2
21p11<>q11
(B)
PKD1
16p13.3
YPKD1
gene
cluster
16p13.11
Y1
Y2
Y3
Y4
Y5
Y6
273
Figure 9.10 The class I HLA gene family: a
clustered gene family with nonprocessed
pseudogenes and gene fragments.
(A) Structure of a class I HLA heavy-chain
mRNA. The full-length mRNA contains a
polypeptide-encoding sequence with a
leader sequence (L), three extracellular
domains (a1, a2, and a3), a transmembrane
sequence (TM), a cytoplasmic tail (CY), and
a 3¢ untranslated region (3¢ UTR). The three
extracellular domains are each encoded
essentially by a single exon. The very small
5¢ UTR is not shown. (B) The class I HLA heavy
chain gene cluster is located at 6p21.3 and
comprises about 20 genes. They include six
expressed genes (ﬁlled blue boxes), four
full-length nonprocessed pseudogenes
(long red open boxes labeled y), and a
variety of partial gene copies (short open red
boxes labeled 1–7). Some of the latter are
truncated at the 5¢ end (e.g. 1, 3, 5, and 6),
some are truncated at the 3¢ end (e.g. 7), and
some contain single exons (e.g. 2 and 4).
Figure 9.11 Dispersal of nonprocessed
NF1 and PKD1 pseudogenes as a result
of pericentromeric or subtelomeric
instability. (A) The NF1 neuroﬁbromatosis
type I gene is located close to the
centromere of human chromosome 17. It
spans 283 kb and has 58 exons. Exons are
represented by thin vertical boxes; introns
are shown by connecting chevrons (^).
Highly homologous defective copies of the
NF1 gene are found at nine or more other
genome locations, mostly in pericentromeric
regions. Each copy has a portion of the fulllength gene, with both exons and introns.
Seven examples are shown here, such as
two copies on 15p that have intact genomic
sequences spanning exons 13 and 27b.
Rearrangements have sometimes caused
the deletion of exons and introns (shown
by asterisks). (B) The 46 kb PKD1 polycystic
kidney disease gene is located close to the
telomere of 16p and has over 40 exons. As
a result of segmental duplication events
during primate evolution, large components
of this gene have been duplicated and six
PKD1 pseudogenes are located at 16p13.11,
with large blocks of sequence (shown as blue
boxes) copied from the PKD1 gene (asterisks
represent the absence of counterparts to
PKD1 sequences).
274
Chapter 9: Organization of the Human Genome
(A)
(B)
E1
E2
E3
5¢
3¢
P
transcription and
RNA processing
5¢
AAAA
TTTT
integration in
chromosomal DNA
An 3¢ mRNA
reverse
transcriptase
3¢
5¢
3¢
TTTTTN¢n
TTTT
AAAAANn
TTTTT
3¢
5¢
second-strand synthesis
and DNA repair
Tn 5¢ cDNA
5¢
3¢
9.3
3¢
5¢
AAAAANn
TTTTTN¢n
AAAAANn
TTTTTN¢n
AAAA
TTTT
A AAAAANn
Tn TTTTTN¢n
3¢
5¢
RNA GENES
Much of the attention paid to human genes has focused on protein-coding genes
because they were long considered to be by far the functionally most important
part of our genome. By comparison, genes whose ﬁnal products are functional
noncoding RNA (ncRNA) molecules have been so underappreciated that one of
the two draft human genome sequences reported in 2001 contained no analyses
at all of human RNA genes! RNA was seen to be important in very early evolution
(Box 9.3) but its functions were imagined to have been very largely overtaken by
DNA and proteins. In recent times, the vast majority of RNA molecules were
imagined to serve as accessory molecules in the making of proteins.
The last few years have witnessed a revolution in our understanding of the
importance of RNA and, although the number of protein-encoding genes has
been steadily revised downward since draft human genome sequences were
reported in 2001, the number of RNA genes is constantly being revised upward.
The tiny mitochondrial genome was always considered to be exceptional because
65% (24 out of 37) of its genes are RNA genes. Now we are beginning to realize
that the RNA transcribed from the nucleus is not so uniformly dedicated to protein synthesis as we once thought; instead, it shows great functional diversity.
What has changed our thinking? First, completely unsuspected classes of
ncRNA have recently been discovered, including several proliﬁc classes of tiny
regulatory RNAs. Second, recent whole genome analyses using microarrays and
high-throughput transcript sequencing have shown that at least 85% and possibly more than 90% of the human genome is transcribed. That is, more than 85%
of the nucleotide positions in the euchromatic genome are represented in primary transcripts produced from at least one of the two DNA strands. Two other
major surprises were the extent of multigenic transcription and the pervasiveness of bidirectional transcription. The recent data challenge the distinction
between genes and intergenic space and have forced a radical rethink of the concept of a gene (Box 9.4).
Figure 9.12 Processed pseudogenes
and retrogenes originate by reverse
transcription from RNA transcripts.
(A) In this example, a protein-coding gene
with three exons (E1–E3) is transcribed from
an upstream promoter (P), and introns are
excised from the transcript to yield an mRNA.
The mRNA can then be converted naturally
into an antisense single-stranded cDNA by
using cellular reverse transcriptase function
(provided by LINE-1 repeats). (B) Integration
of the cDNA is envisaged at staggered
breaks (indicated by curly arrows) in A-rich
sequences, but could be assisted by the
LINE-1 endonuclease. If the A-rich sequence
is included in a 5¢ overhang, it could form
a hybrid with the distal end of the poly(T)
of the cDNA, facilitating second-strand
synthesis. Because of the staggered breaks
during integration, the inserted sequence
will be ﬂanked by short direct repeats
(boxed sequences).
TABLE 9.8 EXAMPLES OF HUMAN INTRONLESS RETROGENES AND THEIR PARENTAL INTRONCONTAINING HOMOLOGS
Retrogene
Intron-containing homolog
Product
GK2 at 4q13
GK1 at Xp21
glycerol kinase
PDHA2 at 4q22
PDHA1 at Xp22
pyruvate dehydrogenase
PGK2 at 6p12
PGK at Xq13
phosphoglycerate kinase
TAF1L at 9p13
TAF1 at Xq13
TATA box binding protein associated factor, 250 kD
MYCL1 at 1p34
MYCL2 at Xq22
homolog of v-Myc oncogene
GLUD1 at 10q23
GLUD2 at Xq25
glutamate dehydrogenase
RBMXL at 9p13
RBMX at Xq26
RNA-binding protein important in brain development
RNA GENES
BOX 9.3 THE RNA WORLD HYPOTHESIS
Proteins cannot self-replicate, and so many evolutionary geneticists consider that autocatalytic
nucleic acids must have pre-dated proteins and were able to replicate without the help of
proteins. The RNA world hypothesis developed from ideas proposed by Alexander Rich and
Carl Woese in the 1960s. It imagines that RNA had a dual role in the earliest stages of life, acting
as both the genetic material (with the capacity for self-replication) and also as eﬀector molecules.
Both roles are still evident today: some viruses have RNA genomes, and noncoding RNA molecules
can work as eﬀector molecules with catalytic activity. RNAse P, for example, is a ribozyme that
can cleave substrate RNA without any requirement for protein, and certain types of intron
are autocatalytic and able to splice themselves out of RNA transcripts without any help from
proteins (see text). Another observation consistent with RNA’s being the ﬁrst nucleic acid is that
deoxyribonucleotides are synthesized from ribonucleotides in cellular pathways.
As well as storing genetic information, RNA has been imagined to have been used
subsequently to synthesize proteins from amino acids. Diﬀerent RNAs, including rRNA and
tRNA, are central in assisting polypeptide synthesis. Many ribosomal proteins can be deleted
without aﬀecting ribosome function, and the crucial peptidyl transferase activity—the enzyme
that catalyzes the formation of peptide bonds—is a ribozyme. However, RNA has a rather rigid
backbone and so is not very well suited as an eﬀector molecule. Proteins are much more ﬂexible
and also oﬀer more functional variety because the 20 amino acids can have widely diﬀerent
structures and oﬀer more possible sequence combinations (a decapeptide provides 2010 or about
1013 diﬀerent possible amino acid sequences, whereas a decanucleotide has 410 or about 106
diﬀerent possible sequences).
The replacement of RNA with DNA as an information storage molecule provided signiﬁcant
advantages. DNA is much more stable than RNA, and so is better suited for this task. Its sugar
residues lack the 2¢ OH group on ribose sugars that makes RNA prone to hydrolytic cleavage.
Greater eﬃciency could be achieved by separating the storage and transmission of genetic
information (DNA) from protein synthesis (RNA). All that was needed was the development of a
reverse transcriptase so that DNA could be synthesized from deoxynucleotides by using an RNA
template.
We have known for many decades that various ubiquitous ncRNA classes are
essential for cell function. Until recently, however, we have largely been accustomed to thinking of ncRNA as not much more than a series of accessories that are
needed to process genes to make proteins. Transfer RNAs are needed at the very
end of the pathway, serving to decode the codons in mRNA and provide amino
acids in the order they are needed for insertion into growing polypeptide chains.
Ribosomal RNAs are essential components of the ribosomes, the complex ribonucleoprotein factories of protein synthesis.
Other ubiquitous ncRNAs were known to function higher up the pathway to
ensure the correct processing of mRNA, rRNA, and tRNA precursors. Various
small RNAs are components of complex ribonucleoproteins involved in different
processing reactions, including splicing, cleavage of rRNA and tRNA precursors,
and base modiﬁcations that are required for RNA maturation. Typically these
RNAs work as guide RNAs, by base pairing with complementary sequences in the
precursor RNA.
We have also long been aware of a few ncRNAs that have other functions, such
as RNAs implicated in X-inactivation and imprinting, and the RNA component of
the telomerase ribonucleoprotein needed for synthesis of the DNA of telomeres
(see Figure 2.13). But these RNAs seemed to be quirky exceptions.
In the past decade or so, however, there has been a revolution in how we view
RNA. Many thousands of different ncRNAs have recently been identiﬁed in animal cells. Many of them are developmentally regulated and have been shown to
have crucial roles in a whole variety of different processes that occur in specialized tissues or speciﬁc stages of development. Several ncRNAs have already been
implicated in cancer and genetic disease.
Now that the human genome is known to have close to 20,000 protein-coding
genes, about the same as in the 1 mm nematode Caenorhabditis elegans, which
has only about 1000 cells—the question now is whether RNA-based regulation is
the key feature in explaining our complexity. Certainly, the complexity of RNAbased gene regulation increases markedly in complex organisms, as is described
in Chapter 10. Maybe it is time to view the genome as more of an RNA machine
than just a protein machine.
275
276
Chapter 9: Organization of the Human Genome
BOX 9.4 REVISING THE CONCEPT OF A GENE IN THE POSTGENOME ERA
noncoding DNA sequences than had been expected. And so it
Until genome-wide analyses were performed, a typical human
proved. An unexpectedly high number of conserved regulatory
protein-coding gene was imagined to be well deﬁned and separated
sequences and numerous novel noncoding RNAs (ncRNAs) have
from its neighbors by identiﬁable intergenic spaces. The gene would
been, and continue to be, identiﬁed, often within or spanning
typically be split into several exons. Directed from an upstream
introns of known protein-coding genes.
promoter, a primary transcript complementary to just one DNA strand
As a result of intensive analyses of mammalian transcriptomes,
would undergo splicing. The functionally unimportant (junk) intronic
many thousands of transcripts of unknown function have been
sequences would be discarded, allowing fusion of the important
uncovered. Typically, ncRNA and protein-coding transcripts overlap,
exonic sequences to make an mRNA. Expression would be regulated
creating complicated patterns of transcription (Figure 2). In some
by nearby regulatory sequences typically located close to the
promoter.
cases, such as the imprinted SNURF–SNRPN transcript at 15q12, a
This neat and cosy image of a gene has been buﬀeted by a series
single transcript contains a coding RNA plus a noncoding RNA that are
of complications. It has long been known that some nuclear genes
separated by RNA cleavage.
partly overlap others or are entirely embedded
within much larger genes. Diﬀerent products
gene 1
gene 2
gene 4
were known to be produced from a single gene
by using alternative promoters, alternative
5¢
3¢
splicing, and RNA editing. Very occasionally,
3¢
5¢
sequences from diﬀerent genes could be
gene 3
spliced together at the RNA level, sometimes
even in the case of genes on diﬀerent
annotated exons
chromosomes (trans-splicing). Occasionally,
5¢
natural antisense transcripts were observed
novel TARs
that could be seen to regulate the expression
of the sense transcript of a gene. Some genes
were known to have regulatory elements
forward
located many hundreds of kilobases away,
strand
sometimes within another gene.
transcripts
Despite the above complications, scientists
were not quite ready to give up on the simple
idea of a gene described in the ﬁrst paragraph
above, until whole genome analyses shattered
3¢
previous misconceptions. The signiﬁcant
ﬁndings that forced a reappraisal of gene
reverse
3¢
organization were:
strand
• Transcription is pervasive. More than 85%
transcripts
5¢
of the euchromatic human genome is
transcribed, and multigenic transcription is
Figure 1 Blurring of gene boundaries at the transcript level. In the past, the four genes
common, so that the distinction between
at the top would be expected to behave as discrete non-overlapping transcription units.
genes and intergenic space is now much
As shown by recent analyses, the reality is more complicated. A variety of transcripts often
less apparent (Figure 1). About 70% of
links exons in neighboring genes. The transcripts frequently include sequences from
human genes are transcribed from both
previously unsuspected transcriptionally active regions (TARs). [From Gerstein MB, Bruce C,
strands.
Rozowsky JS et al. (2007) Genome Res. 17, 669–681. With permission from Cold Spring Harbor
• Coding DNA accounts for less than oneLaboratory Press.]
quarter of the highly conserved (and
presumably functionally important) fraction
of the genome. This meant that there must
be many more functionally important
(A)
5¢
3¢
Figure 2 Extensive transcriptional
complexity of human genes. (A) Human
genes are frequently transcribed on both
strands, as shown in this hypothetical gene
cluster. (B) A single gene can have multiple
transcriptional start sites (right-angled arrows)
as well as many interleaved coding and
noncoding transcripts. Exons are shown as
blue boxes. Known short RNAs such as small
nucleolar RNAs (snoRNAs) and microRNAs
(miRNAs) can be processed from intronic
sequences, and novel species of short RNAs
that cluster around the beginning and end
of genes have recently been discovered (see
the text). [From Gingeras TR (2007) Genome
Res. 17, 682–690. With permission from Cold
Spring Harbor Laboratory Press.]
3¢
5¢
(B)
short RNAs
snoRNA
ncRNA
short RNAs
miRNA
protein-coding RNA
5¢
3¢
3¢
5¢
antisense
ncRNA
short RNAs
antisense
ncRNA
short RNAs
RNA GENES
protein
synthesis
and export
RNA
maturation
DNA
synthesis
specific
splicing
regulator
snRNA
cleavage
mRNA
TERC
general
transcription
regulation
HBII-52
snoRNA
long RNAs in
cis-antisense
regulation
U1 snRNA
of pre-tRNA
Y RNA
RNase P
tRNA
of pre-rRNA
7SL RNA
transposon
control
gene
regulation
splicing
rRNA
277
base
modification
RNase
MRP
U2 snRNA
piRNA
endo-siRNA
TSIX
SRA1 RNA
AIR
7SK RNA
RNase MRP
epigenetic
gene
RNAi-based
gene silencing regulation
of rRNA
of snRNA
snoRNA
scaRNA
miRNA
XIST
endo-siRNA
H19
long RNAs as
trans-acting
regulators
HOTAIR
HBII-85
snoRNA
Figure 9.13 Functional diversity of RNA. Various ubiquitous RNAs function in housekeeping roles in cells, and in protein synthesis
and protein export from cells (using 7SL RNA, the RNA component of the signal recognition particle). RNAs involved in RNA
maturation include spliceosomal small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small Cajal body RNAs (scaRNAs),
and two RNA ribonucleases. Telomere DNA synthesis is performed by a ribonucleoprotein that consists of TERC (the telomerase RNA
component) and a reverse transcriptase (see Figure 2.13). The Y RNA family are involved in chromosomal DNA replication, and RNase
MRP has a crucial role in initiating mtDNA replication as well as in cleaving pre-rRNA precursors in the nucleolus. Diverse classes of
RNA serve a regulatory function in gene expression. Although some do have general accessory roles in transcription, regulatory
RNAs are often restricted in their expression to certain cell types and/or developmental stages. Three classes of tiny RNA use RNA
interference (RNAi) pathways to act as regulators: Piwi protein-interacting RNAs (piRNAs) regulate the activity of transposons in
germ-line cells; microRNAs (miRNAs) regulate the expression of target genes; and endogenous short interfering RNAs (endo-siRNAs)
act as gene regulators and also regulate some types of transposon. Some members of the snoRNA family, such as HBII-85 snoRNA,
are involved in epigenetic gene regulation. A large number of long RNAs are involved in regulating various genes, often at the
transcriptional level. Some are known to be involved in epigenetic gene regulation in imprinting, X-inactivation, and so on. RNA
families are shown in black type; individual RNAs are shown in red.
Figure 9.13 gives a modern perspective on the functional diversity of RNA. In
this section we consider the functions and gene organizations of the different
human RNA classes (Table 9.9). Numerous databases have been developed
recently to document data on ncRNAs (Table 9.10).
More than 1000 human genes encode an rRNA or tRNA, mostly
within large gene clusters
Ribosomal RNA genes
In addition to the two mitochondrial rRNA molecules (12S and 16S rRNA), there
are four types of cytoplasmic rRNA, three associated with the large ribosome subunit (28S, 5.8S, and 5S rRNAs) and one with the small ribosome subunit (18S
rRNA). The 5S rRNA genes occur in small gene clusters, the largest being a cluster
of 16 genes on chromosome 1q42, close to the telomere. Only a few 5S rRNA
genes have been validated as functional, and there are many dispersed
pseudogenes.
The 28S, 5.8S, and 18S rRNAs are encoded by a single multigenic transcription
unit (see Figure 1.22) that is tandemly repeated to form megabase-sized
ribosomal DNA arrays (about 30–40 tandem repeats, or roughly 100 rRNA genes)
on the short arms of each of the acrocentric human chromosomes 13, 14, 15, 21,
and 22. We do not know the precise gene numbers because the ribosomal DNA
arrays were excluded from the Human Genome Project, as a result of the technical difﬁculties in obtaining unambiguous ordering of overlapping DNA clones for
long regions composed of very similar tandem repeats.
278
Chapter 9: Organization of the Human Genome
TABLE 9.9 MAJOR CLASSES OF HUMAN NONCODING RNA
RNA class
Subclass or
evolutionary/
functional
subfamily
No. of
diﬀerent
types
Function
Gene organization, biogenesis, etc.
Ribosomal
RNA (rRNA),
~120–5000
nucleotides
12S rRNA, 16S rRNA
1 of each
components of mitochondrial ribosomes
cleaved from multigenic transcripts produced
by H strand of mtDNA (Figure 9.3)
5S rRNA, 5.8S rRNA,
18S rRNA, 28S rRNA
1 of each
components of cytoplasmic ribosomes
5S rRNA is encoded by multiple genes in
various gene clusters; 5.8S, 18S, and 28S
rRNA are cleaved from multigenic transcripts
(Figure 1.22); the multigenic 5.8S–18S–28S
transcription units are tandemly repeated on
each of 13p,14p,15p, 21p, and 22p (= rDNA
clusters)
Transfer
RNA (tRNA),
~70–80
nucleotides
mitochondrial family
22
decode mitochondrial mRNA to make 13
proteins on mitochondrial ribosomes
single-copy genes. tRNAs are cleaved from
multigenic mtDNA transcripts (Figure 9.3)
cytoplasmic family
49
decode mRNA produced by nuclear genes
(Figure 9.13)
700 tRNA genes and pseudogenes dispersed
at multiple chromosomal locations with some
large gene clusters
spliceosomal family
with subclasses Sm
and Lsm (Table 9.10)
9
U1, U2, U4, U5, and U6 snRNAs process
standard GU–AG introns (Figure 1.19);
U4atac, U6atac, U11, and U12 snRNAs
process rare AU–AC introns
about 200 spliceosomal snRNA genes are
found at multiple locations but there are
moderately large clusters of U1 and U2 snRNA
genes; most are transcribed by RNA pol II
non-spliceosomal
snRNAs
several
U7 snRNA: 3¢ processing of histone mRNA;
7SK RNA: general transcription regulator;
Y RNA family: involved in chromosomal
DNA replication and regulators of cell
proliferation
mostly single-copy functional genes
C/D box class
(Figure 9.15A)
246
maturation of rRNA, mostly nucleotide
site-speciﬁc 2¢-O-ribose methylations
H/ACA class
(Figure 9.15B)
94
maturation of rRNA by modifying uridines
at speciﬁc positions to give pseudouridine
usually within introns of protein-coding
genes; multiple chromosomal locations, but
some genes are found in multiple copies in
gene clusters (such as the HBII-52 and HBII-85
clusters—Figure 11.22)
Small Cajal
body RNA
(scaRNA)
25
maturation of certain snRNA classes in
Cajal bodies (coiled bodies) in the nucleus
usually within introns of protein-coding
genes
RNA
ribonucleases,
~260–320
nucleotides
2
RNase P cleaves pre-tRNA in nucleus +
mitochondria; RNase MRP cleaves rRNA
in nucleolus and is involved in initiating
mtDNA replication
single-copy genes
BC200
1
neural RNA that regulates dendritic
protein biosynthesis; originated from Alu
repeat
1 gene, BCYRN1, at 2p16
7SL RNA
3
component of the signal recognition
particle (SRP) that mediates insertion of
secretory proteins into the lumen of the
endoplasmic reticulum
three closely related genes clustered on
14q22
TERC (telomerase
RNA component)
1
component of telomerase, the
ribonucleoprotein that synthesizes
telomeric DNA, using TERC as a template
(Figure 2.13)
single-copy gene at 3q26
Vault RNA
3
components of cytoplasmic vault RNPs
that have been thought to function in
drug resistance
VAULTRC1, VAULTRC2, and VAULTRC3 are
clustered at 5q31 and share ~84% sequence
identity
Y RNA
4
components of the 60 kD Ro
ribonucleoprotein, an important target of
humoral autoimmune responses
RNY1, RNY3, RNY4, and RNY5 are clustered at
7q36
Small nuclear
RNA (snRNA),
~60–360
nucleotides
Small
nucleolar RNA
(snoRNA),
~60–300
nucleotides
Miscellaneous
small
cytoplasmic
RNAs,
~80–500
nucleotides
RNA GENES
279
TABLE 9.9 cont. MAJOR CLASSES OF HUMAN NONCODING RNA
RNA class
Subclass or
evolutionary/
functional
subfamily
No. of
diﬀerent
types
Function
Gene organization, biogenesis, etc.
MicroRNA
(miRNA),
~22 nucleotides
> 70 families of
related miRNAs
~1000
multiple important roles in gene
regulation, notably in development,
and implicated in some cancers
see Figure 9.17 for examples of genome
organization, and Figure 9.16 for how they are
synthesized
Piwi-binding RNA
(piRNA), ~24–31
nucleotides
89 individual
clusters
> 15,000
often derived from repeats; expressed
only in germ-line cells, where they
limit excess transposon activity
89 large clusters distributed across the genome;
individual clusters span from 10 kb to 75 kb
with an average of 170 piRNAs per cluster
Endogenous
short interfering
RNA (endosiRNA), ~21–22
nucleotides
many
probably
more than
10,000a
often derived from pseudogenes,
inverted repeats, etc.; involved in
gene regulation in somatic cells and
may also be involved in regulating
some types of transposon
clusters at many locations in the genome
Long noncoding
regulatory RNA,
often > 1 kb
many
> 3000
involved in regulating gene
expression; some are involved
in monoallelic expression
(X-inactivation, imprinting), and/or as
antisense regulators (Table 9.11)
usually individual gene copies; transcripts often
undergo capping, splicing, and polyadenylation
but antisense regulatory RNAs are typically long
transcripts that do not undergo splicing
aBased on extrapolation of studies in mouse cells.
Transfer RNA genes
The 22 different mitochondrial tRNAs are made by 22 tRNA genes in mtDNA. The
Genomic tRNA Database lists over 500 human tRNA genes that make a cytoplasmic tRNA with a deﬁned anticodon speciﬁcity. The genes can be classiﬁed into 49
families on the basis of anticodon speciﬁcity (Box 9.5). There is only a rough correlation of human tRNA gene number with amino acid frequency. For example,
30 tRNA genes specify the comparatively rare amino acid cysteine (which
accounts for 2.25% of all amino acids in human proteins), but only 21 tRNA genes
specify the more abundant proline (which has a frequency of 6.10%).
Although the tRNA genes seem to be dispersed throughout the human genome,
more than half of human tRNA genes (273 out of 516) reside on either chromosome 6 (with many clustered in a 4 Mb region at 6p2) or chromosome 1. In addition, 18 of the 30 Cys tRNAs are found in a 0.5 Mb stretch of chromosome 7.
TABLE 9.10 MAJOR NONCODING RNA DATABASES
Database
Description
URL
NONCODE
integrated database of all ncRNAs except rRNA and tRNA
http://www.noncode.org
Noncoding RNA database
sequences and functions of noncoding transcripts
http://biobases.ibch.poznan.pl/ncRNA/
RNAdb
comprehensive mammalian noncoding RNA database
http://research.imb.uq.edu.au/rnadb/
Rfam
noncoding RNA families and sequence alignments
http://rfam.sanger.ac.uk/
antiCODE
natural antisense transcripts database
http://www.anticode.org
sno/scaRNAbase
small nucleolar RNAs and small Cajal body-speciﬁc RNAs
http://gene.fudan.sh.cn/snoRNAbase.nsf
snoRNA-LBME-db
comprehensive human snoRNAs
http://www-snorna.biotoul.fr/
Genomic tRNA Database
comprehensive tRNA sequences
http://lowelab.ucsc.edu/GtRNAdb/
Compilation of tRNA sequences
and sequences of tRNA genes
just as its name suggests
http://www.tRNA.uni-bayreuth.de
miRBase
miRNA sequences and target genes
http://microrna.sanger.ac.uk/
piRNAbank
empirically known sequences and other related information
on piRNAs reported in various organisms, including human,
mouse, rat, and Drosophila
http://pirnabank.ibab.ac.in/
280
Chapter 9: Organization of the Human Genome
BOX 9.5 ANTICODON SPECIFICITY OF EUKARYOTIC CYTOPLASMIC tRNAs
There is no one-to-one correspondence between codons in cytoplasmic mRNA and the tRNA
anticodons that recognize them. The 64 possible codons are shown in Figure 1, alongside the
(unmodiﬁed) anticodons. Horizontal lines join codon–anticodon pairs. Alternative codons that
diﬀer in having a C or a U at the third base position can be recognized by a single anticodon (thirdbase wobble, shown by chevrons). There are three rules for decoding cytoplasmic mRNA codons:
• Codons in two-codon boxes. Those codons ending with U/C that encode a diﬀerent amino acid
from those ending with A/G are known as two-codon boxes. Here the U/C wobble position
is typically decoded by a G at the 5¢ base position in the tRNA anticodon. For example, at the
top left for Phe, there is no tRNA with an AAA anticodon to match the UUU codon, but the
GAA anticodon can recognize both UUU and UUC codons in the mRNA (see Figure 1).
• Non-glycine codons in four-codon boxes. Four-codon boxes are those in which U, C, A, and G
in the third, wobble, position all encode the same amino acid. Here the U/C wobble position
is decoded by a chemically modiﬁed adenosine, known as inosine, at the 5¢ position in the
anticodon (blue shaded boxes; see Figure 11.31 for the structure of inosine). Inosine can
base pair with A, C, or U. For example, at the bottom left, the GUU and GUC codons of the
four-codon valine box are decoded by a tRNA with an anticodon of AAC, which is no doubt
modiﬁed to IAC. The IAC anticodon can recognize each of GUU, GUC, and GUA. To avoid
possible translational misreading, tRNAs with inosine at the 5¢ base of the anticodon cannot
be used in two-codon boxes.
• Glycine codons. The four-codon glycine box provides the one exception to the rule above:
GGU and GGC codons are decoded by a GCC anticodon, rather than the expected ICC
anticodon.
Thus, only 16 anticodons are required to decode the 32 codons ending in a U/C. The minimum
set of anticodons is therefore 45 (64 minus 3 stop codons, minus 16). On this basis, one would
predict a total of 45 diﬀerent classes of human tRNA. However, despite the generality of third-base
wobble, three pairs of codons ending in U/C are served by two anticodons each (see Figure 1), and
so there are an extra three tRNA classes. In addition, a specialized tRNA carries an anticodon to
the codon UGA (which normally functions as a stop codon). At high selenium concentrations, this
tRNA will very occasionally decode UGA to insert the 21st amino acid, selenocysteine, in a select
group of selenoproteins. Thus, there are 45 + 3 + 1 = 49 diﬀerent classes of human tRNA, encoded
by several hundred genes (see Figure 1).
UUU
UUC
UUA
Leu
UUG
AAA GAA 12
UAA 7
CAA 7
UCU
UCC
Ser
UCA
UCG
AGA 11
GGA UGA 5
CGA 4
UAU
UAC
stop UAA
stop UAG
AUA 1
GUA 14
UUA CUA -
CUU
CUC
CUA
CUG
AAG 12
GAG UAG 3
CAG 10
Pro
CCU
CCC
CCA
CCG
AGG 10
GGG UGG 7
CGG 4
CAU
CAC
CAA
Gln
CAG
AUG GUG 11
UUG 11
CUG 20
AUU
Ile AUC
AUA
Met AUG
AAU 14
GAU 3
UAU 5
CAU 20
ACU
ACC
Thr
ACA
ACG
AGU 10
GGU UGU 6
CGU 6
AAU
AAC
AAA
Lys
AAG
AUU 2
GUU 32
UUU 16
CUU 17
GUU
GUC
GUA
GUG
AAC 11
GAC UAC 5
CAC 16
Ala
GCU
GCC
GCA
GCG
AGC 29
GGC UGC 9
CGC 5
GAU
GAC
GAA
Glu
GAG
AUC GUC 19
UUC 13
CUC 13
Phe
Leu
Val
Tyr
His
Asn
Asp
UGU
UGC
stop UGA
Trp UGG
Cys
ACA GCA 30
UCA -(3)
CCA 9
CGU
CGC
CGA
CGG
ACG
GCG
UCG
CCG
7
6
4
AGU
AGC
AGA
Arg
AGG
ACU
GCU
UCU
CCU
8
6
5
Arg
Ser
Gly
GGU
GGC
GGA
GGG
ACC GCC 15
UCC 9
CCC 7
Dispersed gene families make various small nuclear RNAs that
facilitate general gene expression
Various families of rather small RNA molecules (60–360 nucleotides long) are
known to have a role in the nucleus in assisting general gene expression, mostly
at the level of post-transcriptional processing. Initially, such RNAs were simply
labeled as small nuclear RNAs (snRNAs) to distinguish them from pre-mRNA.
Many of them were known to be uridine-rich and they were named accordingly
(U2 snRNA, for example, does not honor a famous Irish rock band but simply
indicates the second uridine-rich small nuclear RNA to be classiﬁed). Like rRNA,
Figure 1 Over 500 diﬀerent human
cytoplasmic tRNAs decode the 61
codons that specify the standard 20
amino acids. The relationships between
the 64 possible codons (positioned next
to amino acids on the left of the four
major columns) and the corresponding
anticodons (to the right of the four
columns) are shown. The number next to
each anticodon is the number of diﬀerent
human tRNAs that are documented in the
Genomic tRNA Database (see Table 9.9)
as carrying that anticodon. Note that 12
of the 61 anticodons that could recognize
the codons that specify the standard 20
amino acids are not represented in the
tRNAs (shown by dashes). This happens
because of wobble at the third base
position of most codons where the third
base is U or C (exceptions are for codon
pairs AUU/AUC, AAU/AAC, and UAU/
UAC). The (3) indicated by an asterisk
signiﬁes that there are three diﬀerent
selenocysteine tRNAs with an anticodon
that can recognize the codon UGA,
which normally serves as a stop codon.
The shaded adenines are most probably
a modiﬁed form of adenine known
as inosine, in which the amino group
attached to carbon 6 is replaced by a
C=O carbonyl group.
RNA GENES
281
TABLE 9.11 THE Sm AND Lsm CLASSES OF SPLICEOSOMAL snRNA
Sm classa
Lsm class
Component of major spliceosome
U1, U2, U4, and U5 snRNAs
U6 snRNA
Component of minor spliceosome
U11, U12, U4atac, and U5 snRNAs
U6atac snRNA
Structure
see Figure 9.14A
see Figure 9.14B
Transcribed by
RNA polymerase II
RNA polymerase III
Bound core proteins
Sm proteins (SmB, SmD1, SmD2, SmD3, SmE, SmF, SmG)
seven Lsm proteins (LSM2–LSM8)
Location
synthesized in the nucleus and then exported to the
cytoplasm, where they each associate with seven Sm
proteins and undergo 5¢ and 3¢ end processing; then
re-imported into the nucleus to undergo more RNA
processing in Cajal bodies, before accumulating in
speckles to perform spliceosomal function
never leave the nucleus; undergo maturation in
the nucleolus and then accumulate in speckles to
perform spliceosomal function
Site-speciﬁc nucleotide
modiﬁcation
performed by scaRNAs in the Cajal bodies of the nucleus
performed by snoRNAs in the nucleolus
aAlthough not a spliceosomal snRNA, U7 snRNA has a similar structure to the Sm class of snRNAs and ﬁve of its seven core proteins are identical to core
proteins bound by the Sm snRNAs.
snRNA molecules bind various proteins and function as ribonucleoproteins
(snRNPs).
Subsequently, various snRNAs, including some of the ﬁrst to be classiﬁed,
were found to be involved in post-transcriptional processing of rRNA precursors
in the nucleolus; they were therefore re-classiﬁed as small nucleolar RNAs
(snoRNAs), for example U3 and U8 snoRNAs. More recently, membership of the
classes has been based on structural and functional classiﬁcation.
A third group of small RNAs have been identiﬁed that resemble snoRNAs but
are conﬁned to Cajal bodies (also called coiled bodies), discrete nuclear structures in the nucleus that are closely associated with the maturation of snRNPs.
They have been termed small Cajal body RNAs (scaRNAs). Hundreds of mostly
dispersed human genes are devoted to making snRNA and snoRNA, and there are
many hundreds of associated pseudogenes.
(A)
Spliceosomal small nuclear RNA (snRNA) genes
The nine human spliceosomal snRNAs vary in length from 106 to 186 nucleotides
and bind a ring of seven core proteins. U1, U2, U4, U5, and U6 snRNAs operate
within the major spliceosome to process conventional GU–AG introns (see Figure
1.19). U4atac, U6atac, U11, and U12 snRNAs form part of the minor spliceosome
that excises rare AU–AC introns. Each of the spliceosomal snRNPs contains seven
core proteins that are identical within a subclass and a unique set of snRNPspeciﬁc proteins. The Lsm subclass is made up of just U6 and U6atac snRNAs; the
other spliceosomal snRNAs belong to the Sm subclass (Table 9.11 and Figure
9.14).
More than 70 genes specify snRNAs used in the major spliceosome. They
include 44 identiﬁed genes specifying U6 snRNA and 16 specifying U1 snRNA.
There is some evidence for clustering. Multiple U2 snRNA genes are found at
17q21–q22 but the copy number varies; a cluster of about 30 U1 snRNA genes is
located at 1p36.1.
Figure 9.14 Structures of Sm-type and Lsm-type spliceosomal snRNAs.
(A) Sm-type snRNAs contain three important recognition elements: a
5¢-trimethylguanosine (TMG) cap, an Sm-protein-binding site (Sm site), and
a 3¢ stem–loop structure. The Sm site and the 3¢ stem elements are required
for recognition by the survival motor neuron (SMN) complex for assembly
into stable core ribonucleoproteins (RNPs). The consensus Sm site directs the
assembly of a ring of the seven Sm core proteins (see Table 9.10). The TMG cap
and the assembled Sm core proteins are required for recognition by the nuclear
import machinery. (B) Lsm-type snRNAs contain a 5¢-monomethylphosphate
guanosine (MPG) cap and a 3¢ stem, and terminate in a stretch of uridine
residues (the Lsm site) that is bound by the seven Lsm core proteins.
CH3
CH3
3¢ stem
GpppApNp
CH3
OCH3
Sm site
TMG cap
consensus
AAUUUUUGG
(B)
3¢ stem
CH3
pppGp
Lsm site
MPG cap
UUUUU
282
Chapter 9: Organization of the Human Genome
Non-spliceosomal small nuclear RNA genes
Not all snRNAs within the nucleoplasm function as part of spliceosomes. Both
U1 and U2 snRNAs also have non-spliceosomal functions. U1 snRNA is required
to stimulate transcription by RNA polymerase II. U2 snRNA is known to stimulate
transcriptional elongation by RNA polymerase II. Several other small nuclear
RNAs with a non-spliceosomal function have been well studied. They tend to be
single-copy genes but there are many associated pseudogenes. Three examples
are given below.
• U7 snRNA is a 63-nucleotide snRNA that is dedicated to the specialized 3¢
processing undergone by histone mRNA which, exceptionally, is not
polyadenylated.
•
7SK RNA is a 331-nucleotide RNA that functions as a negative regulator of the
RNA polymerase II elongation factor P-TEFb.
•
The Y RNA family consists of three small RNAs (less than 100 nucleotides long)
that are involved in chromosomal DNA replication and function as regulators
of cell proliferation.
Small nucleolar RNA (snoRNA) genes
SnoRNAs are between 60 and 300 nucleotides long and were initially identiﬁed in
the nucleolus, where they guide nucleotide modiﬁcations in rRNA at speciﬁc
positions. They do this by forming short duplexes with a sequence of the rRNA
that contains the target nucleotide. There are two large subfamilies. H/ACA
snoRNAs guide site-speciﬁc pseudouridylations (uridine is isomerized to give
pseudouridine at 95 different positions in the pre-rRNA). C/D box snoRNAs guide
site-speciﬁc 2¢-O-ribose methylations (there are 105–107 varieties of this methylation in rRNA). Single snoRNAs specify one, or at most two, such base modiﬁcations (Figure 9.15).
At least 340 human snoRNA genes have been found so far, but there may be
many more because snoRNAs are surprisingly difﬁcult to identify with the use of
bioinformatic approaches. The vast majority are found within the introns of
larger genes that are transcribed by RNA polymerase II. These snoRNAs are produced by processing of the intronic RNA, and so the regulation of their synthesis
is coupled to that of the host gene. Many snoRNA genes are dispersed single-copy
genes. Others occur in clusters. For example, the large imprinted SNURF–SNRPN
transcription unit at 15q12 contains six different types of C/D box snoRNA gene,
two of which are present in large gene clusters: one contains about 45 almost
identical HBII-52 snoRNA genes and the other 29 HBII-85 snoRNA genes (see
Figure 11.22).
Most snoRNAs are ubiquitously expressed, but some are tissue-speciﬁc. For
example, the six types of snoRNA gene within the SNURF–SNRPN transcription
unit are predominantly expressed in the brain from only the paternal chromo(A)
3¢
5¢
u
g
a box C¢
u
g
u
a
box D¢ g
u
c
(B)
m
3¢
m
A
G
U
A
box C G
U
R
NY
N
NY
C
U box D
G
A
5¢
3¢
cap
P
OH
5¢
cap
P
box H
ANANNA
box ACA
ACANNNOH
Figure 9.15 Structure and function of
snoRNAs. (A) C/D box snoRNAs guide
2¢-O-methylation modiﬁcations. The box
C and D motifs and a short 5¢, 3¢-terminal
stem formed by intrastrand base pairing
(shown as a series of short horizontal red
lines) constitute a kink-turn structural motif
that is speciﬁcally recognized by the
15.5 kD snoRNP protein. The C¢ and D¢ boxes
represent internal, frequently imperfect
copies of the C and D boxes. C/D box
snoRNAs and their substrate RNAs form a
10–21 bp double helix in which the target
residue to be methylated (shown here by
the letter m in a circle) is positioned exactly
ﬁve nucleotides upstream of the D or D¢
box. R represents purine. (B) H/ACA box
snoRNAs guide the conversion of uridines
to pseudouridine. These RNAs fold into a
hairpin–hinge–hairpin–tail structure. One
or both of the hairpins contains an internal
loop, called the pseudouridylation pocket,
that forms two short (3–10 bp) duplexes with
nucleotides ﬂanking the unpaired substrate
uridine (y) located about 15 nucleotides
from the H or ACA box of the snoRNA.
Although each box C/D and H/ACA snoRNA
could potentially direct two modiﬁcation
reactions, apart from a few exceptions, most
snoRNAs possess only one functional
2¢-O-methylation or pseudouridylation
domain.
RNA GENES
some 15. Nonstandard functions are known or expected for some snoRNA genes
that do not have sequences complementary to rRNA sequences. For example, the
HBII-52 snoRNA has an 18-nucleotide sequence that is perfectly complementary
to a sequence within the HTR2C (serotonin receptor 2c) gene at Xp24, and
regulates alternative splicing of this gene. The neighboring HBII-85 snoRNAs
have recently been implicated in the pathogenesis of Prader–Willi syndrome
(OMIM 176270).
Small Cajal body RNA genes
The scaRNAs resemble snoRNAs and perform a similar role in RNA maturation,
but their targets are spliceosomal snRNAs and they perform site-speciﬁc modiﬁcations of spliceosomal snRNA precursors in the Cajal bodies of the nucleus.
There are at least 25 human genes, each specifying one type of scaRNA. Like
snoRNA genes, the scaRNA genes are typically located within the introns of genes
transcribed by RNA polymerase II.
Close to 1000 diﬀerent human microRNAs regulate complex sets
of target genes by base pairing to the RNA transcripts
In addition to tRNA, we have known for some time about a variety of other moderately small (80–500-nucleotide) cytoplasmic RNAs. For example, the enzyme
telomerase that synthesizes DNA at telomeres (see Figure 2.13) has both a protein component, TERT (telomerase reverse transcriptase), and also an RNA component, TERC, that is synthesized by a single-copy gene at 3q26.2 (see Table 9.8
for other examples). In the early 2000s it became clear that a novel family of tiny
regulatory RNAs known as microRNA (miRNA) also operated in the cytoplasm.
MicroRNAs are only about 21–22 nucleotides long on average, and they were
initially missed in analyses of the human genome. The ﬁrst animal miRNAs to be
reported were identiﬁed in model organisms such as the nematode (C. elegans)
and the fruit ﬂy (Drosophila melanogaster) by investigators studying phenomena
relating to RNA interference (Box 9.6), a natural form of gene regulation that
protects cells from harmful propagation of viruses and transposons.
Because many miRNAs are strongly conserved during evolution, vertebrate
miRNAs were quickly identiﬁed and the ﬁrst human miRNAs were reported in
the early 2000s. miRNAs regulate the expression of selected sets of target genes by
base pairing with their transcripts. Usually, the binding sites are in the 3¢ untranslated region of target mRNA sequences, and the bound miRNA inhibits translation so as to down-regulate expression of the target gene.
Synthesis of miRNAs involves the cleavage of RNA precursors by nucleusspeciﬁc and cytoplasm-speciﬁc RNase III ribonucleases, nucleases that speciﬁcally bind to and cleave double-stranded RNAs. The primary transcript, the
pri-miRNA, has closely positioned inverted repeats that base-pair to form a hairpin RNA that is initially cleaved from the primary transcript by a nuclear RNase
III (known as Rnasen or Drosha) to make a short double-stranded pre-miRNA
that is transported out of the nucleus (Figure 9.16). A cytoplasmic RNase III called
dicer cleaves the pre-miRNA to generate a miRNA duplex with overhanging
3¢ dinucleotides.
A speciﬁc RNA-induced silencing complex (RISC) that contains the endoribonuclease argonaute binds the miRNA duplex and acts so as to unwind the double-stranded miRNA. The argonaute protein then degrades one of the RNA
strands (the passenger strand) to leave the mature single-stranded miRNA (known
as the guide strand) bound to argonaute. The mature miRNP associates with RNA
transcripts that have sequences complementary to the guide strand. The binding
of miRNA to target transcript normally involves a signiﬁcant number of base mismatches. As a result, a typical miRNA can silence the expression of hundreds of
target genes in much the same way that a tissue-speciﬁc protein transcription
factor can affect the expression of multiple target genes at the same time—see
the targets section of the miRBase database listed in Table 9.9.
To identify more miRNA genes, new computational bioinformatics programs
were developed to screen genome sequences. By mid-2009, more than 700 human
miRNA genes had been identiﬁed and experimentally validated, but comparative
genomics analyses indicate that the number of such genes is likely to increase.
Some of the miRNA genes have their own individual promoters; others are part of
283
284
Chapter 9: Organization of the Human Genome
BOX 9.6 RNA INTERFERENCE AS A CELL DEFENSE MECHANISM
RNA interference (RNAi) is an evolutionarily ancient mechanism that is
used in animals, plants, and even single-celled fungi to protect cells
against viruses and transposable elements. Both viruses and active
transposable elements can produce long double-stranded RNA, at
least transiently during their life cycles. Long double-stranded RNA is
not normally found in cells and, for many organisms, it triggers an RNA
interference pathway. A cytoplasmic endoribonuclease called dicer
cuts the long RNA into a series of short double-stranded RNA pieces
known as short interfering RNA (siRNA). The siRNA produced is on
average 21 bp long, but asymmetric cutting produces two-nucleotide
overhangs at their 3¢ ends (Figure 1).
The siRNA duplexes are bound by diﬀerent complexes that
contain an argonaute-type endoribonuclease (Ago) and some other
proteins. Thereafter, the two RNA strands are unwound and one of
the RNA strands is degraded by argonaute, leaving a single-stranded
RNA bound to the argonaute complex. The argonaute complex is now
activated; the single-stranded RNA will guide the argonaute complex
to its target by base-pairing with complementary RNA sequences in
the cells.
One type of argonaute complex is known as the RNA-induced
silencing complex (RISC). In this case, after the single-stranded guide
RNA binds to a complementary long single-stranded RNA, the
argonaute enzyme will cleave the RNA, causing it to be degraded. Viral
and transposon RNA can be inactivated in this way.
Another class of argonaute complex is the RNA-induced
transcriptional silencing (RITS) complex. Here, the single-stranded RNA
guide binds to complementary RNA transcripts as they emerge during
transcription by RNA polymerase II. This allows the RITS complex
to position itself on a speciﬁc part of the genome and then attract
proteins such as histone methyltransferases (HMTs) and sometimes
DNA methyltransferases (DNMTs), which covalently modify histones in
the immediate region. This process eventually causes heterochromatin
formation and spreading; in some cases, the RITS complex can
induce DNA methylation. As a result, gene expression can be
silenced over long periods to limit, for example, the activities
of transposons.
Although mammalian cells have RNA interference pathways,
the presence of double-stranded RNA triggers an interferon response
that causes nonspeciﬁc gene silencing and cell death. This is
described in Chapter 12 when we consider using RNA interference
as an experimental tool to produce the speciﬁc silencing of preselected target genes. In such cases, artiﬁcially synthesized short
double-stranded RNA is used to trigger RNAi-based gene
silencing.
long ds RNA
dicer
Ago + other
RISC proteins
Ago + other
RITS proteins
siRNA
activated RITS
activated RISC
cap
poly(A)
cap
poly(A)
cap
poly(A)
DNA
HMT
DNMT
histone methylation
DNA methylation
degraded RNA
transcription repressed
Figure 1 RNA interference. Long double-stranded (ds) RNA is cleaved by cytoplasmic
dicer to give siRNA. siRNA duplexes are bound by argonaute complexes that unwind the
duplex and degrade one strand to give an activated complex with a single RNA strand. By
base pairing with complementary RNA sequences, the siRNA guides argonaute complexes
to recognize target sequences. Activated RISC complexes cleave any RNA strand that is
complementary to their bound siRNA. The cleaved RNA is rapidly degraded. Activated
RITS complexes use their siRNA to bind to any newly synthesized complementary RNA
and then attract proteins, such as histone methyltransferases (HMT) and sometimes DNA
methyltransferases (DNMT), that can modify the chromatin to repress transcription.
RNA GENES
285
a miRNA cluster and are cleaved from a common multi-miRNA transcription
unit (Figure 9.17A). Another class of miRNA genes form part of a compound
transcription unit that is dedicated to making other products in addition to
miRNA, either another type of ncRNA (Figure 9.17B) or a protein (Figure 9.17C).
Many thousands of diﬀerent piRNAs and endogenous siRNAs
suppress transposition and regulate gene expression
The discovery of miRNAs was unexpected, but later it became clear that miRNAs
represent a small component of what are a huge number of different tiny regulatory RNAs made in animal cells. In mammals, two additional classes of tiny regulatory RNA were ﬁrst reported in 2006, and these are being intensively studied.
Because huge numbers of different varieties of these RNAs are generated from
multiple different locations in the genome, large-scale sequencing has been
required to differentiate them.
Piwi-protein-interacting RNA
Piwi-protein-interacting RNAs (piRNAs) have been found in a wide variety of
eukaryotes. They are expressed in germ-line cells in mammals and are typically
24–31 nucleotides long; they are thought to have a major role in limiting transposition by retrotransposons in mammalian germ-line cells, but they may also regulate gene expression in some organisms. Control of transposon activity is
required because by integrating into new locations in the genome, active transposons can interfere with gene function, causing genetic diseases and cancer.
(A)
nucleus
miR
RNA pol II
Figure 9.16 Human miRNA synthesis. (A) General scheme. The primary transcript, pri-miRNA, has a 5¢ cap
(m7GpppG) and a 3¢ poly(A) tail. miRNA precursors have a prominent double-stranded RNA structure (RNA
hairpin), and processing occurs through the actions of a series of ribonuclease complexes. In the nucleus,
Rnasen, the human homolog of Drosha, cleaves the pri-miRNA to release the hairpin RNA (pre-miRNA); this
is then exported to the cytoplasm, where it is cleaved by the enzyme dicer to produce a miRNA duplex.
The duplex RNA is bound by an argonaute complex and the helix is unwound, whereupon one strand (the
passenger) is degraded by the argonaute ribonuclease, leaving the mature miRNA (the guide strand) bound
to argonaute. miR, miRNA gene. (B) A speciﬁc example: the synthesis of human miR-26a1. Inverted repeats
(shown as highlighted sequences overlined by long arrows) in the pri-miRNA undergo base pairing to form
a hairpin, usually with a few mismatches. The sequences that will form the mature guide strand are shown in
red; those of the passenger strand are shown in blue. Cleavage by both the human Drosha and dicer (green
arrows) is typically asymmetric, leaving an RNA duplex with overhanging 3¢ dinucleotides.
pri-miRNA
(B)
cap
poly(A)
cap
GUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGUGCAGGUCCCAAUGGGCCUAUUCUUGGUUACUUGCACGGGGACGC
hairpin formation
RNASEN complex
cap
pre-miRNA
AAAAA
c a
g
u
u
g
g
gug ccucgu caaguaa ccaggauaggcugu
g
u
cgc ggggca guucauu gguucuauccggua a
c c c
a
c
-
pri-miRNA
RNASEN complex
cytoplasm
c a
u
u
g
g
u caaguaa ccaggauaggcugu
g
u
gca guucauu gguucuauccggua a
c c c
c
-
pre-miRNA
DICER complex
DICER complex
5¢
miRNA duplex
ARGONAUTE complex
miRNA
pre-miRNA
u
u
u caaguaa ccaggauaggcu 3¢
3¢ gca guucauu gguucuaucc
c
-
miRNA duplex
5¢
ARGONAUTE complex
uucaaguaauccaggauaggcu
miRNA
AAAAA
286
Chapter 9: Organization of the Human Genome
(A)
miR-21
cap
poly(A)
miR-17~18~19a~20~19b–1~92–1
cap
poly(A)
(B)
miR-155 in BIC ncRNA
cap
poly(A)
miR-15a~16-1 in DLEU2 ncRNA intron
cap
poly(A)
(C)
miR-198 in FSTL1 mRNA 3¢ UTR
cap
poly(A)
miR-106b~93~25 in MCM7 pre-mRNA intron
cap
hairpin RNA
poly(A)
exonic
coding RNA
exonic
noncoding RNA
intronic RNA
More than 15,000 different human piRNAs have been identiﬁed and so the
piRNA family is among the most diverse RNA family in human cells (see Table
9.8). The piRNAs map back to 89 genomic intervals of about 10–75 kb long (for
more information see the piRNAbank database; Table 9.9). They are thought to be
cleaved from large multigenic transcripts. In humans, the multi-piRNA transcripts contain sequences for up to many hundreds of different piRNAs.
piRNAs are thought to repress transposition by transposons through an RNA
interference pathway, by association with piwi proteins, which are evolutionarily
related to argonaute proteins (Figure 9.18).
Endogenous siRNAs
Long double-stranded RNA in mammalian cells triggers nonspeciﬁc gene silencing through interferon pathways, but transfection of exogenous synthetic siRNA
duplexes or short hairpin RNAs induces RNAi-mediated silencing of speciﬁc
genes with sequence elements in common with the exogenous RNA. As we will
see in Chapter 12, this is an extremely important experimental tool that can give
valuable information on the cellular functions of a gene. Very recently, it has
become clear that human cells also naturally produce endogenous siRNAs (endosiRNAs).
In mammals, the most comprehensive endo-siRNA analyses have been performed in mouse oocytes. Like piRNAs, endo-siRNAs are among the most varied
RNA population in the cell (many tens of thousands of different endo-siRNAs
have been identiﬁed in mouse oocytes). They arise as a result of the production
of limited amounts of natural double-stranded RNA in the cell. One way in
which this happens involves the occasional transcription of some pseudogenes
(Figure 9.19).
Figure 9.17 The structure of human primiRNAs. (A) Examples of transcripts that are
used exclusively to make miRNAs: miR-21
is produced from a single hairpin within a
dedicated primary transcript RNA;
a single multigenic transcript with six
hairpins that will eventually be cleaved to
give six miRNAs, namely miR-17, miR-18,
miR-19a, and so on. (B, C) Examples of
miRNAs that are co-transcribed with a gene
encoding either (B) a long noncoding RNA
(ncRNA) or (C) a polypeptide. In each part,
the upper example shows single miRNAs
located within (B) an exon of an ncRNA
(miR-155) and (C) in the 3¢ untranslated
region (UTR) within a terminal exon of an
mRNA (miR-198). The lower examples show
multiple miRNAs located within intronic
sequences of (B) an ncRNA (miR-15a and
miR-16-1) and (C) a pre-mRNA (miR-106b,
miR-93, and miR-25). Cap, m7G(5¢)ppp(5¢)
G. [Adapted from Du T & Zamore PD
(2005) Development 132, 4645–4652. With
permission from the Company of Biologists.]
RNA GENES
Figure 9.18 piRNA-based transposon
silencing in animal cells. (A) Primary
piRNAs (piwi-protein-interacting RNAs) are
24–31 nucleotides long and are processed
from long RNA precursors transcribed
from deﬁned loci called piRNA clusters.
Any transposon inserted in the reverse
orientation in the piRNA cluster can give rise
to antisense piRNAs (shown in red).
(B) Antisense piRNAs are incorporated into
a piwi protein and direct its slicer activity on
sense transposon transcripts. The 3¢ cleavage
product is bound by another piwi protein
and trimmed to piRNA size. This sense piRNA
is, in turn, used to cleave piRNA cluster
transcripts and to generate more antisense
piRNAs. (C) Antisense piRNAs target the piwi
complexes to cDNA for DNA methylation
(left) and/or histone modiﬁcation (right).
DNMT, DNA methyltransferase; HMT, histone
methyltransferase; HP1, heterochromatin
protein 1. [From Girard A & Hannon GJ (2007)
Trends Cell Biol. 18, 136–148. With permission
from Elsevier.]
(A) detection
primary piRNAs
sense
transposon
antisense
transposon
?
piRNA cluster
(B) amplification
loaded piwi
protein
transposon
transcript
cleaved
transposon
transcript
piwi
piwi
piwi
piwi
piRNA cluster
transcript
DNA
methylation
(in mammals)
histone
modifications
(C) repression
piwi
DNMT
Me
piwi
Me Me
Me Me
HP1
HP1
287
HMT
More than 3000 human genes synthesize a wide variety of
medium-sized to large regulatory RNAs
Many thousands of different long ncRNAs, often many kilobases in length, are
also thought to have regulatory roles in animal cells. They include antisense transcripts that usually do not undergo splicing and that can regulate overlapping
sense transcripts, plus a wide variety of long mRNA-like ncRNAs that undergo
capping, splicing, and polyadenylation but do not seem to encode any sizeable
polypeptide, although some contain internal ncRNAs such as snoRNAs and
piRNAs. The functions of the great majority of the mRNA-like ncRNAs are
unknown. Some, however, are known to be tissue-speciﬁc and involved in gene
regulation. Recently, in a systematic effort to identify long ncRNAs, 3300 different
human long ncRNAs were identiﬁed as associating with chromatin-modifying
complexes, thereby affecting gene expression.
Some long mRNA-like ncRNAs that are involved in epigenetic regulation have
been extensively studied. The XIST gene encodes a long ncRNA that regulates
X-chromosome inactivation, the process by which one of the two X chromosomes
is randomly selected to be condensed in female mammals, with large regions
becoming transcriptionally inactive. Many other long ncRNAs, such as the H19
RNA, are implicated in repressing the transcription of either the paternal or
maternal allele of many autosomal regions (imprinting). These mRNA-like
ncRNAs are often regulated by genes that produce what can be very long antisense ncRNA transcripts that usually do not undergo splicing (Table 9.12 gives
examples).
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Chapter 9: Organization of the Human Genome
nucleus
RNA
A
retrotransposition
duplication
duplication
and
inversion
yA
* *
* *
yA
* *
yA
A
5¢
cytoplasm
mRNA
5¢
antisense transcript
+
5¢
hairpin
dicer
21-nucleotide
siRNA
Figure 9.19 Pseudogenes can regulate
the expression of their parent gene
by endogenous siRNA pathways.
Pseudogenes arise through the copying
of a parent gene. Some pseudogenes are
transcribed and, depending on the genomic
context, can produce an RNA that is the
antisense equivalent of the mRNA produced
by the parent gene. An mRNA transcript
of the parent gene (A) and an antisense
transcript of a corresponding pseudogene
(yA) can then form a double-stranded
RNA that is cleaved by dicer to give siRNA.
Endogenous siRNAs can also be produced
from duplicated inverted sequences such
as the example shown here of an inverted
duplication of the pseudogene (yA yA) at
the right. Transcription through both copies
of the pseudogene results in a long RNA with
inverted repeats (blue, overlined arrows)
causing the RNA to fold into a hairpin that
is cleaved by dicer to give siRNA. In either
case, the endogenous siRNAs are guided
by RISC to interact with, and degrade, the
parent gene’s remaining mRNA transcripts.
Green arrows indicate DNA rearrangements.
[Adapted from Sasidharan R & Gerstein M
(2008) Nature 453, 729–731. With permission
from Macmillan Publishers Ltd.]
RISC
mRNA cleavage
The pervasive involvement of long ncRNAs in regulating developmental processes is illustrated by a comprehensive (5 bp resolution) analysis of the transcriptional output at the four human HOX gene clusters. Although there are only 39
HOX genes, the transcriptional output of the HOX clusters was also found to
include a total of 231 different long ncRNAs. Many of these are cis-acting regulators, but one of them, HOTAIR, was found to be a trans-acting regulator (see Table
9.12).
Some of the functional RNAs, such as XIST and AIR, have not been so well
conserved during evolution. The fastest-evolving functional sequences in the
human genome include components of primate-speciﬁc long ncRNAs that are
strongly expressed in brain. We consider the evolutionary implications of such
genes in Chapter 10.
TABLE 9.12 EXAMPLES OF LONG REGULATORY HUMAN RNAs
RNA
Size
Gene location
Gene organization
Function
XIST
19.3 kb
Xq13
6 exons spanning 32 kb
regulator of X-chromosome inactivation
TSIX
37.0 kb
Xq13
1 exon
antisense regulator of XIST
H19
2.3 kb
11p15
5 exons spanning 2.67 kb
involved in imprinting at the 11p15 imprinted cluster
associated with Beckwith–Wiedemann syndrome
KCNQTOT1 (= LIT1)
59.5 kb
11p15
1 exon
antisense regulator at the imprinted cluster at 11p15
PEG3
1.8 kba
19q13
variable number of exons but
up to 9 exons spanning 25 kb
maternally imprinted and known to function in tumor
suppression by activating p53
HOTAIR
2.2 kb
12q13
6 exons spanning 6.3 kb
trans-acting gene regulator; although part of a
regulatory region in the HOX-C cluster on 12q13, HOTAIR
RNA represses transcription of a 40 kb region on the
HOX-D cluster on chromosome 2q31
aLargest isoforms.
HIGHLY REPETITIVE DNA: HETEROCHROMATIN AND TRANSPOSON REPEATS
289
9.4 HIGHLY REPETITIVE DNA: HETEROCHROMATIN
AND TRANSPOSON REPEATS
Genes contain some repetitive DNA sequences, including repetitive coding DNA.
However, the majority of highly repetitive DNA sequences occur outside genes.
Some of the sequences are present at certain subchromosomal regions as large
arrays of tandem repeats. This type of DNA, known as heterochromatin, remains
highly condensed throughout the cell cycle and does not generally contain
genes.
Other highly repetitive DNA sequences are interspersed throughout the
human genome and were derived by duplicative transposition (see Section 9.1).
Sequences like this are sometimes described as transposon repeats and they
account for more than 40% of the total DNA sequence in the human genome. In
addition to residing in extragenic regions, they are often found in introns and
untranslated sequences and sometimes even in coding sequences.
Constitutive heterochromatin is largely deﬁned by long arrays of
high-copy-number tandem DNA repeats
The DNA of constitutive heterochromatin accounts for 200 Mb or 6.5% of the
human genome (see Table 9.3). It encompasses megabase regions at the centromeres and comparatively short lengths of DNA at the telomeres of all chromosomes. Most of the Y chromosome and most of the short arms of the acrocentric
chromosomes (13, 14, 15, 21, and 22) consist of heterochromatin. In addition,
there are very substantial heterochromatic regions close to the centromeres of
certain chromosomes, notably chromomosomes 1, 9, 16, and 19.
The DNA of constitutive heterochromatin mostly consists of long arrays of
high-copy-number tandemly repeated DNA sequences, known as satellite DNA
(Table 9.13). Shorter arrays of tandem repeats are known as minisatellites and
TABLE 9.13 MAJOR CLASSES OF HIGHCOPYNUMBER TANDEMLY REPEATED HUMAN DNA
Classa
Total array size
unit
Satellite DNAb
often hundreds of
kilobases
Size or sequence of
repeat unit
Major chromosomal location(s)
associated with heterochromatin
a (alphoid DNA)
171 bp
centromeric heterochromatin of all chromosomes
b (Sau3A family)
68 bp
notably the centromeric heterochromatin of 1, 9, 13, 14, 15,
21, 22, and Y
Satellite 1
25–48 bp (AT-rich)
centromeric heterochromatin of most chromosomes and
other heterochromatic regions
Satellite 2
diverged forms of
ATTCC/GGAAT
most, possibly all, chromosomes
Satellite 3
ATTCC/GGAAT
13p, 14p, 15p, 21p, 22p, and heterochromatin on 1q, 9q,
and Yq12
DYZ19
125 bp
~400 kb at Yq11
DYZ2
AT-rich
Yq12; higher periodicity of ~2470 bp
Minisatellite DNA
0.1–20 kb
at or close to telomeres of all chromosomes
Telomeric minisatellite
TTAGGG
all telomeres
Hypervariable minisatellites
9–64 bp
all chromosomes, associated with euchromatin, notably in
sub-telomeric regions
often 1–4 bp
widely dispersed throughout all chromosomes
Microsatellite DNA
< 100 bp
aThe distinction between satellite, minisatellite, and microsatellite is made on the basis of the total array length, not the size of the repeat unit.
bSatellite DNA arrays that consist of simple repeat units often have base compositions that are radically diﬀerent from the average 41% G+C
(and so could be isolated by buoyant density gradient centrifugation, when they would be diﬀerentiated from the main DNA and appear as satellite
bands—hence the name).
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Chapter 9: Organization of the Human Genome
microsatellites, respectively. Large tracts of heterochromatin are typically composed of a mosaic of different satellite DNA sequences that are occasionally interrupted by transposon repeats but are devoid of genes. Transposon repeats are
also widely distributed in euchromatin and will be described below.
The vast majority of human heterochromatic DNA has not been sequenced,
because of technical difﬁculties in obtaining unambiguous ordering of overlapping DNA clones. Thus, for example, only short representative components of
centromeric DNA have been sequenced so far. However, Y-chromosome heterochromatin is an exception and has been well characterized. There are different
satellite DNA organizations, and the repeated unit may be a very simple sequence
(less than 10 nucleotides long) or a moderately complex one that can extend to
over 100 nucleotides long; see Table 9.13.
At the sequence level, satellite DNA is often extremely poorly conserved
between species. Its precise function remains unclear, although some human
satellite DNAs are implicated in the function of centromeres whose DNA consists
very largely of various families of satellite DNA.
The centromere is an epigenetically deﬁned domain. Its function is independent of the underlying DNA sequence; instead, its function depends on its particular chromatin organization, which, once established, has to be stably maintained
through multiple cell divisions. Of the various satellite DNA families associated
with human centromeres, only the a-satellite is known to be present at all human
centromeres, and its repeat units often contain a binding site for a speciﬁc centromere protein, CENPB. Cloned a-satellite arrays have been shown to seed de
novo centromeres in human cells, indicating that a-satellite must have an important role in centromere function.
The specialized telomeric DNA consists of medium-sized arrays just a few
kilobases long and constitutes a form of minisatellite DNA. Unlike satellite DNA,
telomeric minisatellite DNA has been extraordinarily conserved during vertebrate evolution and has an integral role in telomere function. It consists of arrays
of tandem repeats of the hexanucleotide TTAGGG that are synthesized by the
telomerase ribonucleoprotein (see Figure 2.13).
Transposon-derived repeats make up more than 40% of the
human genome and arose mostly through RNA intermediates
Almost all of the interspersed repetitive noncoding DNA in the human genome is
derived from transposons (also called transposable elements), mobile DNA
sequences that can migrate to different regions of the genome. Close to 45% of
the genome can be recognized as belonging to this class, but much of the remaining unique DNA must also be derived from ancient transposon copies that have
diverged extensively over long evolutionary time-scales.
In humans and other mammals there are four major classes of transposon
repeat, but only a tiny minority of transposon repeats are actively transposing.
According to the method of transposition, the repeats can be organized into two
groups:
• Retrotransposons (also abbreviated to retroposons). Here the copying mechanism resembles the way in which processed pseudogenes and retrogenes are
generated (see Figure 9.12): reverse transcriptase converts an RNA transcript
of the retrotransposon into a cDNA copy that then integrates into the genomic
DNAs at a different location. Three major mammalian transposon classes use
this copy-and-paste mechanism: long interspersed nuclear elements (LINEs),
short interspersed nuclear elements (SINEs), and retrovirus-like elements
containing long terminal repeats.
• DNA transposons. Members of this fourth class of transposon migrate directly
without any copying of the sequence; the sequence is excised and then reinserted elsewhere in the genome (a cut-and-paste mechanism).
Transposable elements that can transpose independently are described as
autonomous; those that cannot are known as nonautonomous (Figure 9.20). Of
the four classes of transposable element, LINEs and SINEs predominate; we
describe them more fully below. The other two classes are brieﬂy described
here.
HIGHLY REPETITIVE DNA: HETEROCHROMATIN AND TRANSPOSON REPEATS
LINEs
LINE-1 family
LINE-2 family
LINE-3 family
~600,000
~370,000
~44,000
autonomous
P
ORF1 ORF2(pol)
~1,200,000
~450,000
~85,000
retrovirus-like (LTR transposons)
HERV families
MaLR
(A)n
6–8 kb
SINEs
Alu family
MIR
MIR3
nonautonomous
~240,000
~285,000
(A)n
100–400 bp
LTR
P
LTR
gag pol (env)
LTR (gag) LTR
6–11 kb
1.5–3 kb
DNA transposon fossils
MER1 (Charlie)
~213,000
MER2 (Tigger)
~68,000
others
~60,000
(including marine, etc.)
transposase
2–3 kb
80 bp–3 kb
Human LTR transposons
LTR transposons include autonomous and nonautonomous retrovirus-like elements that are ﬂanked by long terminal repeats (LTRs) containing necessary
transcriptional regulatory elements. Endogenous retroviral sequences contain gag
and pol genes, which encode a protease, reverse transcriptase, RNAse H, and
integrase. They are thus able to transpose independently. There are three major
classes of human endogenous retroviral sequence (HERV), with a cumulative
copy number of about 240,000, accounting for a total of about 4.6% of the human
genome (see Figure 9.20).
Very many HERVs are defective, and transposition has been extremely rare
during the last several million years. However, the very small HERV-K group
shows conservation of intact retroviral genes, and some members of the HERVK10 subfamily have undergone transposition comparatively recently during evolution. Nonautonomous retrovirus-like elements lack the pol gene and often also
the gag gene (the internal sequence having been lost by homologous recombination between the ﬂanking LTRs). The MaLR family of such elements accounts for
almost 4% or so of the genome.
Human DNA transposon fossils
DNA transposons have terminal inverted repeats and encode a transposase that
regulates transposition. They account for close to 3% of the human genome and
can be grouped into different classes that can be subdivided into many families
with independent origins (see the Repbase database of repeat sequences at
http://www.girinst.org/repbase/index.html). There are two major human families, MER1 and MER2, plus a variety of less frequent families (see Figure 9.20).
Virtually all the resident human DNA transposon sequences are no longer
active; they are therefore transposon fossils. DNA transposons tend to have short
lifespans within a species, unlike some of the other transposable elements such
as LINEs. However, quite a few functional human genes seem to have originated
from DNA transposons, notably genes encoding the RAG1 and RAG2 recombinases and the major centromere-binding protein CENPB.
A few human LINE-1 elements are active transposons and enable
the transposition of other types of DNA sequence
LINEs (long interspersed nuclear elements) have been very successful transposons. They have a comparatively long evolutionary history, occurring in other
mammals, including mice. As autonomous transposons, they can make all the
products needed for retrotransposition, including the essential reverse transcriptase. Human LINEs consist of three distantly related families: LINE-1,
LINE-2, and LINE-3, collectively comprising about 20% of the genome (see Figure
9.20). They are located primarily in euchromatic regions and are located preferentially in the dark AT-rich G bands (Giemsa-positive) of metaphase chromosomes.
291
Figure 9.20 Mammalian transposon
families. Only a small proportion of
members of any of the illustrated transposon
families may be capable of transposing;
many have lost such a capacity after
acquiring inactivating mutations, and many
are short truncated copies. Subclasses of
the four main families are listed, along with
sizes in base pairs. ORF, open reading frame.
[Adapted from International Human Genome
Sequencing Consortium (2001) Nature 409,
860–921. With permission from Macmillan
Publishers Ltd.]
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Chapter 9: Organization of the Human Genome
Of the three human LINE families, LINE-1 (or L1) is the only family that continues to have actively transposing members. LINE-1 is the most important
human transposable element and accounts for a higher fraction of genomic DNA
(17%) than any other class of sequence in the genome.
Full-length LINE-1 elements are more than 6 kb long and encode two proteins: an RNA-binding protein and a protein with both endonuclease and reverse
transcriptase activities (Figure 9.21A). Unusually, an internal promoter is located
within the 5¢ untranslated region. Full-length copies therefore bring with them
their own promoter that can be used after integration in a permissive region of
the genome. After translation, the LINE-1 RNA assembles with its own encoded
proteins and moves to the nucleus.
To integrate into genomic DNA, the LINE-1 endonuclease cuts a DNA duplex
on one strand, leaving a free 3¢ OH group that serves as a primer for reverse transcription from the 3¢ end of the LINE RNA. The endonuclease’s preferred cleavage
site is TTTTØA; hence the preference for integrating into AT-rich regions. AT-rich
DNA is comparatively gene-poor, and so because LINEs tend to integrate into
AT-rich DNA they impose a lower mutational burden, making it easier for their
host to accommodate them. During integration, the reverse transcription often
fails to proceed to the 5¢ end, resulting in truncated, nonfunctional insertions.
Accordingly, most LINE-derived repeats are short, with an average size of 900 bp
for all LINE-1 copies, and only about 1 in 100 copies are full length.
The LINE-1 machinery is responsible for most of the reverse transcription in
the genome, allowing retrotransposition of the nonautonomous SINEs and also
of copies of mRNA, giving rise to processed pseudogenes and retrogenes. Of the
6000 or so full-length LINE-1 sequences, about 80–100 are still capable of transposing, and they occasionally cause disease by disrupting gene function after
insertion into an important conserved sequence.
Alu repeats are the most numerous human DNA elements and
originated as copies of 7SL RNA
SINEs (short interspersed nuclear elements) are retrotransposons about
100–400 bp in length. They have been very successful in colonizing mammalian
genomes, resulting in various interspersed DNA families, some with extremely
high copy numbers. Unlike LINEs, SINEs do not encode any proteins and they
cannot transpose independently. However, SINEs and LINEs share sequences at
their 3¢ end, and SINEs have been shown to be mobilized by neighboring LINEs.
By parasitizing on the LINE element transposition machinery, SINEs can attain
very high copy numbers.
The human Alu family is the most prominent SINE family in terms of copy
number, and is the most abundant sequence in the human genome, occurring on
average more than once every 3 kb. The full-length Alu repeat is about 280 bp
long and consists of two tandem repeats, each about 120 bp in length followed by
a short An/Tn sequence. The tandem repeats are asymmetric: one contains an
internal 32 bp sequence that is lacking in the other (Figure 9.21B). Monomers,
(A) LINE-1 repeat element
5¢ UTR
ORF1
ORF2
3¢ UTR
pA
p40
endonuclease
reverse
transcriptase
(B) Alu repeat element
right monomer
left monomer
AAAAAA
TTTTTT
A
AAAAAA
TTTTTT
B
32 bp
Figure 9.21 The human LINE-1 and Alu
repeat elements. (A) The 6.1 kb LINE-1
element has two open reading frames: ORF1,
a 1 kb open reading frame, encodes p40,
an RNA-binding protein that has a nucleic
acid chaperone activity; the 4 kb ORF2
speciﬁes a protein with both endonuclease
and reverse transcriptase activities. A
bidirectional internal promoter lies within
the 5¢ untranslated region (UTR). At the
other end, there is an An/Tn sequence, often
described as the 3¢ poly(A) tail (pA). The
LINE-1 endonuclease cuts one strand of a
DNA duplex, preferably within the sequence
TTTTØA, and the reverse transcriptase uses
the released 3¢-OH end to prime cDNA
synthesis. New insertion sites are ﬂanked
by a small target site duplication of
2–20 bp (ﬂanking black arrowheads).
(B) An Alu dimer. The two monomers have
similar sequences that terminate in an
An/Tn sequence but diﬀer in size because of
the insertion of a 32 bp element within the
larger repeat. Alu monomers also exist in
the human genome, as do various truncated
copies of both monomers and dimers.
CONCLUSION
containing only one of the two tandem repeats, and various truncated versions
of dimers and monomers are also common, giving a genomewide average of
230 bp.
Whereas SINEs such as the MIR (mammalian-wide interspersed repeat) families are found in a wide range of mammals, the Alu family is of comparatively
recent evolutionary origin and is found only in primates. However, Alu subfamilies of different evolutionary ages can be identiﬁed. In the past 5 million or
so years since the divergence of humans and African apes, only about 5000 copies of the Alu repeat have undergone transposition; the most mobile Alu sequences
are members of the Y and S subfamilies.
Like other mammalian SINEs, Alu repeats originated from cDNA copies of
small RNAs transcribed by RNA polymerase III. Genes transcribed by RNA
polymerase III often have internal promoters, and so cDNA copies of transcripts
carry with them their own promoter sequences. Both the Alu repeat and, independently, the mouse B1 repeat originated from cDNA copies of 7SL RNA, the
short RNA that is a component of the signal recognition particle, using a retrotransposition mechanism like that shown in Figure 9.12. Other SINEs, such as the
mouse B2 repeat, are retrotransposed copies of tRNA sequences.
Alu repeats have a relatively high GC content and, although dispersed mainly
throughout the euchromatic regions of the genome, are preferentially located in
the GC-rich and gene-rich R chromosome bands, in striking contrast to the preferential location of LINEs in AT-rich DNA. However, when located within genes
they are, like LINE-1 elements, conﬁned to introns and the untranslated regions.
Despite the tendency to be located in GC-rich DNA, newly transposing Alu
repeats show a preference for AT-rich DNA, but progressively older Alu repeats
show a progressively stronger bias toward GC-rich DNA.
The bias in the overall distribution of Alu repeats toward GC-rich and, accordingly, gene-rich regions must result from strong selection pressure. It suggests
that Alu repeats are not just genome parasites but are making a useful contribution to cells containing them. Some Alu sequences are known to be actively transcribed and may have been recruited to a useful function. The BCYRN1 gene,
which encodes the BC200 neural cytoplasmic RNA, arose from an Alu monomer
and is one of the few Alu sequences that are transcriptionally active under normal circumstances. In addition, the Alu repeat has recently been shown to act as
a trans-acting transcriptional repressor during the cellular heat shock response.
CONCLUSION
In this chapter, we have looked at the architecture of the human genome. Each
human cell contains many copies of a small, circular mitochondrial genome and
just one copy of the much larger nuclear genome. Whereas the mitochondrial
genome bears some similarities to the compact genomes of prokaryotes, the
human nuclear genome is much more complex in its organization, with only
1.1% of the genome encoding proteins and 95% comprising nonconserved, and
often highly repetitive, DNA sequences.
Sequencing of the human genome has revealed that, contrary to expectation,
there are comparatively few protein-coding genes—about 20,000–21,000 according to the most recent estimates. These genes vary widely in size and internal
organization, with the coding exons often separated by large introns, which often
contain highly repetitive DNA sequences. The distribution of genes across the
genome is uneven, with some functionally and structurally related genes found
in clusters, suggesting that they arose by duplication of individual genes or larger
segments of DNA. Pseudogenes can be formed when a gene is duplicated and
then one of the pair accumulates deleterious mutations, preventing its expression. Other pseudogenes arise when an RNA transcript is reverse transcribed and
the cDNA is re-inserted into the genome.
The biggest surprise of the post-genome era is the number and variety of nonprotein-coding RNAs transcribed from the human genome. At least 85% of the
euchromatic genome is now known to be transcribed. The familiar ncRNAs
known to have a role in protein synthesis have been joined by others that have
roles in gene regulation, including several proliﬁc classes of tiny regulatory RNAs
and thousands of different long ncRNAs. Our traditional view of the genome is
being radically revised.
293
294
Chapter 9: Organization of the Human Genome
In Chapter 10 we describe how the human genome compares with other
genomes, and how evolution has shaped it. Aspects of human gene expression
are elaborated in Chapter 11. Within Chapter 13 we also consider human genome
variation.
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